Background Proper management of patients with Kallmann syndrome (KS) allows them to attain a normal reproductive health. The purpose of this study is to demonstrate the presentation modalities, phenotypes and the modes of inheritance among 32 patients with Kallmann syndrome in Jordan. Recognition of the syndrome allows for prompt proper management and provision of genetic counselling.
Background
Proper management of patients with Kallmann syndrome (KS) allows them to attain a normal reproductive health. The purpose of this study is to demonstrate the presentation modalities, phenotypes and the modes of inheritance among 32 patients with Kallmann syndrome in Jordan. Recognition of the syndrome allows for prompt proper management and provision of genetic counselling.Subjects Over a period of five years (1999–2004), the clinical and inheritance profiles of 26 male and 6 female patients with Kallmann syndrome from 12 families were evaluated at the National Center for Diabetes, Endocrinology and Genetics in Jordan.
Subjects
Over a period of five years (1999–2004), the clinical and inheritance profiles of 26 male and 6 female patients with Kallmann syndrome from 12 families were evaluated at the National Center for Diabetes, Endocrinology and Genetics in Jordan.Results The patients belonged to twelve Jordanian and Palestinian families and their age at presentation ranged from 4 – 46 years. Nine boys aged 4–14 years presented with cryptorchidism and microphallus, all other males presented with delayed puberty, hypogonadism and/or infertility. The main presentation among six female patients was primary amenorrhea. Intrafamilial variability in clinical phenotype was specifically evident for renal abnormalities and sensorineural hearing impairment. Familial KS was diagnosed in 27 patients belonging to five families with the X-linked mode of inheritance and two families with the autosomal recessive mode of inheritance.
Results
The patients belonged to twelve Jordanian and Palestinian families and their age at presentation ranged from 4 – 46 years. Nine boys aged 4–14 years presented with cryptorchidism and microphallus, all other males presented with delayed puberty, hypogonadism and/or infertility. The main presentation among six female patients was primary amenorrhea. Intrafamilial variability in clinical phenotype was specifically evident for renal abnormalities and sensorineural hearing impairment. Familial KS was diagnosed in 27 patients belonging to five families with the X-linked mode of inheritance and two families with the autosomal recessive mode of inheritance.Conclusions (1) the majority of cases in this study represented the X-linked form of KS, which might point to a high prevalence of Kal 1 gene in the population. (2) Genetic counselling helps these families to reach a diagnosis at an early age and to decide about their reproductive options. (3) Children presenting with cryptorchidism and microphallus in our population should be investigated for KS.
Conclusions
(1) the majority of cases in this study represented the X-linked form of KS, which might point to a high prevalence of Kal 1 gene in the population. (2) Genetic counselling helps these families to reach a diagnosis at an early age and to decide about their reproductive options. (3) Children presenting with cryptorchidism and microphallus in our population should be investigated for KS.

Background One of the most common causes of hypogonadotropic hypogonadism is Kallmann syndrome (KS). KS is a genetically heterogeneous condition that affects approximately one in 8000 males and one in 40,000–70,000 females [ 1 - 3 ]. This recent estimate is much higher than the previously estimated prevalence of Kallmann syndrome among males of 1:80,000 [ 4 ]. To our knowledge, no data is available on the incidence of this syndrome in Jordan or in the Arab world. In addition to the sporadic form which is the most common [ 2 ], KS has three modes of inheritance, X-linked, autosomal recessive and autosomal dominant [ 5 ]. The gene responsible for the X-linked form of the disease is KAL1 gene [ 6 - 8 ], and encodes the protein anosmin that is directly responsible for the migration of GnRH neurons and the olfactory nerves from the olfactory system to the hypothalamus [ 5 , 9 - 11 ]. Males usually present in the second decade with delayed puberty and females present with primary amenorrhea. Prepubertal boys may present with microphallus and cryptorchidism [ 12 , 13 ]. Proper management of patients with Kallmann syndrome usually allows them to attain normal reproductive health. The purpose of this paper is to demonstrate the presentation modalities, phenotypes and the modes of inheritance among 32 patients with Kallmann syndrome in Jordan, which may contribute to the recognition of the syndrome and the provision of adequate management and genetic counselling.
Background
One of the most common causes of hypogonadotropic hypogonadism is Kallmann syndrome (KS). KS is a genetically heterogeneous condition that affects approximately one in 8000 males and one in 40,000–70,000 females [ 1 - 3 ]. This recent estimate is much higher than the previously estimated prevalence of Kallmann syndrome among males of 1:80,000 [ 4 ].To our knowledge, no data is available on the incidence of this syndrome in Jordan or in the Arab world.In addition to the sporadic form which is the most common [ 2 ], KS has three modes of inheritance, X-linked, autosomal recessive and autosomal dominant [ 5 ]. The gene responsible for the X-linked form of the disease is KAL1 gene [ 6 - 8 ], and encodes the protein anosmin that is directly responsible for the migration of GnRH neurons and the olfactory nerves from the olfactory system to the hypothalamus [ 5 , 9 - 11 ]. Males usually present in the second decade with delayed puberty and females present with primary amenorrhea. Prepubertal boys may present with microphallus and cryptorchidism [ 12 , 13 ]. Proper management of patients with Kallmann syndrome usually allows them to attain normal reproductive health. The purpose of this paper is to demonstrate the presentation modalities, phenotypes and the modes of inheritance among 32 patients with Kallmann syndrome in Jordan, which may contribute to the recognition of the syndrome and the provision of adequate management and genetic counselling.Subjects & Methods Over a period of five years, thirty-two male and female patients from twelve Jordanian and Palestinian families were referred to the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan for evaluation of hypogonadism among adults, or microphallus among children. Prospective evaluation was performed including pedigree construction (fig  1 ), and complete clinical examination with special emphasis on assessment of anosmia, the presence of mirror image movements (synkinesia) and examination of external genitalia. Figure 1 Pedigrees of 12 families with KS Olfactory testing was performed through a smell identification test designed by our department using odours that can be easily identified among all social classes in the local population. These odours included cinnamon, coffee, camomile, thyme, soap, oriental perfume and tobacco. The specific odoriferous substance was approximated to the nostrils while the patient had his eyes closed. The patient is then asked to name the olfactory stimulus. According to the number of substances identified by smelling, patients were divided into anosmic and hyposmic. Those who failed to identify all odours were categorized as anosmic, while those who identified three or less odours were categorized as hyposmic. Those who identified four to all seven odours were considered normosmic. First-degree relatives of the probands were also questioned and tested for the smell sensation in a similar fashion. The same investigator carried out olfactory testing for all patients and family members. In addition to the assessment of smell sensation among relatives of probands, their answers to specific questions were recorded. These questions addressed the issues of surgery to correct cryptorchidism, administration of testosterone and whether there were any complaints of delayed puberty or infertility. The specific questions and the olfactory testing led to the identification of further cases of KS in the family. Patients were asked to perform a screwdriver motion in one hand while the examiner watches for any similar non-voluntary movement in the other hand to assess mirror image movements. Subjects were also asked to perform alternate supination and pronation of one forearm while the examiner watches for a similar movement in the other arm [ 14 ]. Phallus length was measured and compared to age-matched controls [ 15 ], and the testicle size was estimated using an orchidometer. Neurosensory hearing impairment was assessed by audiometry, renal abnormalities by renal ultrasound and congenital heart defects by echocardiography. All patients were tested for color vision and abnormal eye movements. Complete hormonal evaluation including basal gonadotrophins, sex hormones and gonadotrophin releasing hormone (GnRH) stimulation test were done for 20 patients 16 years and above. Three adult patients were not tested due to loss of contact. GnRH test involved the administration of 100 mcg of GnRH, followed by the assessment of gonadotrophin levels at 0, 15, 30, 45, 60 and 120 minutes. For individuals with no or low response to the test, priming was performed, where GnRH was given for five days, with repetition of assessment of gonadotrophin levels. Seminal fluid analysis was done for male patients aged 16 years and above. Radiological studies included brain, pituitary and olfactory tract magnetic resonance imaging (MRI). The criteria for diagnosis of KS among adults included the presence of anosmia or hyposmia with clinical signs and symptoms of hypogonadism and a testosterone level <100 ng/dl among males 16 years and older, and estradiol level <20 pg/dl among adult females, together with basal low gonadotrophin level. In family II, loss of contact of three brothers hindered testing their hormonal levels. They were, however, included among our patients because clinical examination revealed hypogonadism, anosmia and micropenis. Among prepubertal males, the criteria for diagnosis of KS included presence of microphallus with anosmia/hyposmia, and/or absent olfactory bulbs on MRI. Pedigree analysis was used to establish the modes of inheritance of KS in the familial cases. Inheritance in a family was classified as X-linked if only males were affected in more than one sibship connected by females, or if two or more males were affected in the sibship with a negative family history and with associated synkinesia. Inheritance was classified as autosomal recessive if all affected individuals were members of the same generation and included at least one female.
Subjects & Methods
Over a period of five years, thirty-two male and female patients from twelve Jordanian and Palestinian families were referred to the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan for evaluation of hypogonadism among adults, or microphallus among children.Prospective evaluation was performed including pedigree construction (fig  1 ), and complete clinical examination with special emphasis on assessment of anosmia, the presence of mirror image movements (synkinesia) and examination of external genitalia.Olfactory testing was performed through a smell identification test designed by our department using odours that can be easily identified among all social classes in the local population. These odours included cinnamon, coffee, camomile, thyme, soap, oriental perfume and tobacco. The specific odoriferous substance was approximated to the nostrils while the patient had his eyes closed. The patient is then asked to name the olfactory stimulus. According to the number of substances identified by smelling, patients were divided into anosmic and hyposmic. Those who failed to identify all odours were categorized as anosmic, while those who identified three or less odours were categorized as hyposmic. Those who identified four to all seven odours were considered normosmic. First-degree relatives of the probands were also questioned and tested for the smell sensation in a similar fashion. The same investigator carried out olfactory testing for all patients and family members.In addition to the assessment of smell sensation among relatives of probands, their answers to specific questions were recorded. These questions addressed the issues of surgery to correct cryptorchidism, administration of testosterone and whether there were any complaints of delayed puberty or infertility. The specific questions and the olfactory testing led to the identification of further cases of KS in the family.Patients were asked to perform a screwdriver motion in one hand while the examiner watches for any similar non-voluntary movement in the other hand to assess mirror image movements. Subjects were also asked to perform alternate supination and pronation of one forearm while the examiner watches for a similar movement in the other arm [ 14 ]. Phallus length was measured and compared to age-matched controls [ 15 ], and the testicle size was estimated using an orchidometer. Neurosensory hearing impairment was assessed by audiometry, renal abnormalities by renal ultrasound and congenital heart defects by echocardiography. All patients were tested for color vision and abnormal eye movements. Complete hormonal evaluation including basal gonadotrophins, sex hormones and gonadotrophin releasing hormone (GnRH) stimulation test were done for 20 patients 16 years and above. Three adult patients were not tested due to loss of contact. GnRH test involved the administration of 100 mcg of GnRH, followed by the assessment of gonadotrophin levels at 0, 15, 30, 45, 60 and 120 minutes. For individuals with no or low response to the test, priming was performed, where GnRH was given for five days, with repetition of assessment of gonadotrophin levels. Seminal fluid analysis was done for male patients aged 16 years and above. Radiological studies included brain, pituitary and olfactory tract magnetic resonance imaging (MRI).The criteria for diagnosis of KS among adults included the presence of anosmia or hyposmia with clinical signs and symptoms of hypogonadism and a testosterone level <100 ng/dl among males 16 years and older, and estradiol level <20 pg/dl among adult females, together with basal low gonadotrophin level. In family II, loss of contact of three brothers hindered testing their hormonal levels. They were, however, included among our patients because clinical examination revealed hypogonadism, anosmia and micropenis. Among prepubertal males, the criteria for diagnosis of KS included presence of microphallus with anosmia/hyposmia, and/or absent olfactory bulbs on MRI.Pedigree analysis was used to establish the modes of inheritance of KS in the familial cases. Inheritance in a family was classified as X-linked if only males were affected in more than one sibship connected by females, or if two or more males were affected in the sibship with a negative family history and with associated synkinesia. Inheritance was classified as autosomal recessive if all affected individuals were members of the same generation and included at least one female.Results Over a period of five years (1999–2004), thirty-two patients were prospectively diagnosed with Kallmann syndrome at the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan. The patients belonged to twelve Jordanian and Palestinian families and their ages at presentation ranged between 4 – 46 years. They included 26 males and 6 females with a male/female ratio of 4.34/1. Nine male patients were aged 14 years and younger. The clinical features among male patients are presented in table  (1) , and among female patients in table  (2) . Table 1 Clinical features of 26 males with Kallmann syndrome FAMILY No Age Anosmia/hyposmia Synkinesia Hearing Impairment Renal abnormalities Azoospermia cryptorchidism Micropenis 1 1 46 +/H - + - + + - 2 14 + + + + + + - 3 27 + + - + + + - 4 20 + + - + + + - 5 20 + + - + + + - 6 19 + + - + + + - 7 16 + + - + + + - 8 14 + + - + NA + - 9 9 + + - + NA + + 10 6 + + - - NA - + 11 4 + + - - NA + + II 1 37 + - - - + + + 2 24 + - ND* ND* ND* + + 3 22 + - ND* ND* ND* - + 4 20 + - ND* ND* ND* + + III 1 14 + + - - NA - + 2 10 + + - + NA - + 3 8 + + - + NA - + IV 1 6 + + - - NA + + 2 5 + + - - NA + + V 1 20 +/H - + - + - + 2 19 +/H + + + + - + VI 1 19 + - - + ND + - VII 1 37 + - - - + + + VIII 1 22 + - - + + + + IX 1 20 + - - - oligospermia + + NA: not applicable. ND: not done. *: lost contact H: hyposmia Table 2 Clinical Features of 6 females with Kallmann syndrome FAMILY No Age Anosmia/hyposmia Synkinesia Hearing loss Renal abnormalities Primary Amenorrhea X 1 23 + - - - + 2 21 + - - - + 3 18 + - - - + IX 1 18 +/H - - - + XI 1 30 + - - - + XII 1 18 +/H - - - + H: hyposmia Table 3 Hormonal profile and GnRH stimulation test in adult male patients with KS. FAMILY Age Inheritance Base line LH (mIU/ml) Peak LH (mIU/ml) after GnRH testing Peak LH (mIU/ml) after priming Baseline FSH (mIU/ml) Peak FSH (mIU/ml) after GnRH testing Peak FSH (mIU/ml) after priming Testosterone ng/dl 1 1 46 XR 0.5 2.26 1.09 2.22 60 2 14 XR Undetected 0.35 2.50 0.64 2.39 4.25 Undetected 3 27 XR Undetected 0.90 2.85 0.59 1.58 5.24 Undetected 4 20 XR 0.6 2.90 1.66 3.43 80 5 20 XR 0.8 3.10 2.1 5.6 50 6 19 XR Undetected 0.40 2.88 0.8 2.3 6.4 Undetected 7 16 XR 0.6 3..50 1.35 3.21 50 II 1 37 XR Undetected 0.50 3.25 0.43 2.14 6.34 Undetected 2 24 XR ND ND ND ND ND ND ND 3 22 XR ND ND ND ND ND ND ND 4 20 XR ND ND ND ND ND ND ND V 1 20 XR Undetected 0.45 2.1 0.47 1.65 3.2 Undetected 2 19 XR 0.42 4.36 0.90 5.22 40 VI 1 19 sporadic 0.5 6.2 0.9 10.1 70 VII 1 37 sporadic Undetected 1.9 0.46 3.2 Undetected VIII 1 22 sporadic 0.53 3.25 0.85 4.50 Undetected IX 1 20 AR 0.85 4.25 1.21 6.30 50 ND: not done (lost contact) XR :X-linked recessive AR :autosomal recessive Table 4 Hormonal profile and GnRH stimulation test in adult female patients with KS. FAMILY Age Inheritance Base line LH (mIU/ml) N= Peak LH (mIU/ml) after GnRH testing Peak LH (mIU/ml) after priming Base line FSH (mIU/ml) N= Peak FSH (mIU/ml) After GnRH testing Peak FSH (mIU/ml) after priming Estradiol Pg/ml X 1 23 AR 0.6 7.25 2.6 19.33 5 2 21 AR 0.5 3.5 1.95 6.60 7 3 18 AR 0.6 5.2 1.2 9.5 10 IX 1 18 sporadic 0.8 5.10 2.1 5.6 15 XI 1 30 sporadic 0.7 4.2 1.5 6.2 10 XII 1 18 sporadic Undetected 2.10 2.1 5.8 Undetected AR :autosomal recessive Twenty seven patients were anosmic and five patients were hyposmic (table  1 ). In family V, both affected brothers were hyposmic. Cryptorchidism was found or previously operated on in 19/26 (73%) and microphallus in 17/26 (65%) male patients respectively. All patients included in this study manifested high-arched palate. Renal abnormalities including unilateral renal agenesis, malrotated kidney, and horseshoe kidney were detected in 11/19 (58%)cases with the X-linked form of KS. Two sporadic cases showed renal anomalies. A variable degree of sensorineural hearing impairment was found in 4/19 patients with X-linked KS, and in none of the other mode of inheritance or the sporadic cases. Olfactory MRI revealed olfactory tract agenesis among 19/24 cases for which the investigation was done in the series. Among females diagnosed as KS in this series primary amenorrhea was the main presenting feature. Pedigrees were constructed for all families (figure  1 ). Five cases were sporadic and 27 cases were familial belonging to seven families. Pedigree analysis assigned an X-linked mode of inheritance to 3 families with affected males linked through normal females (families I, II and IV). Family I is the largest family in our series with 11 affected males. Two further families were designated as having the X-linked form of KS because the affected males among these siblings displayed synkinesia with a negative family history (families III and V). Synkinesia has been reported to be associated only with the X-linked form of KS [ 16 ]. However, a recent report identifying the specific gene mutated in autosomal dominant KS pointed out that synkinesia may occur in the autosomal forms of KS [ 17 ]. In families III and V in this report, pedigree analysis strongly pointed to the X-linked form of KS although autosomal recessive inheritance cannot be definitely excluded. Two families were designated as having the autosomal recessive mode of inheritance. One family included affected brother and sister with normal consanguineous parents (family IX), while the other family had 3 affected sisters with normal consanguineous parents (family X). Consanguinity rate among parents of all patients was 83%, with 50% of all marriages being between first cousins.
Results
Over a period of five years (1999–2004), thirty-two patients were prospectively diagnosed with Kallmann syndrome at the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan. The patients belonged to twelve Jordanian and Palestinian families and their ages at presentation ranged between 4 – 46 years. They included 26 males and 6 females with a male/female ratio of 4.34/1. Nine male patients were aged 14 years and younger.The clinical features among male patients are presented in table  (1) , and among female patients in table  (2) .Clinical features of 26 males with Kallmann syndrome1IIIIIIVVVIVIIVIIIIXNA: not applicable.ND: not done.*: lost contactH: hyposmiaClinical Features of 6 females with Kallmann syndromeXIXXIXIIH: hyposmiaHormonal profile and GnRH stimulation test in adult male patients with KS.1IIVVIVIIVIIIIXND: not done (lost contact)XR :X-linked recessiveAR :autosomal recessiveHormonal profile and GnRH stimulation test in adult female patients with KS.AR :autosomal recessiveTwenty seven patients were anosmic and five patients were hyposmic (table  1 ). In family V, both affected brothers were hyposmic.Cryptorchidism was found or previously operated on in 19/26 (73%) and microphallus in 17/26 (65%) male patients respectively. All patients included in this study manifested high-arched palate. Renal abnormalities including unilateral renal agenesis, malrotated kidney, and horseshoe kidney were detected in 11/19 (58%)cases with the X-linked form of KS. Two sporadic cases showed renal anomalies. A variable degree of sensorineural hearing impairment was found in 4/19 patients with X-linked KS, and in none of the other mode of inheritance or the sporadic cases. Olfactory MRI revealed olfactory tract agenesis among 19/24 cases for which the investigation was done in the series.Among females diagnosed as KS in this series primary amenorrhea was the main presenting feature.Pedigrees were constructed for all families (figure  1 ). Five cases were sporadic and 27 cases were familial belonging to seven families. Pedigree analysis assigned an X-linked mode of inheritance to 3 families with affected males linked through normal females (families I, II and IV). Family I is the largest family in our series with 11 affected males. Two further families were designated as having the X-linked form of KS because the affected males among these siblings displayed synkinesia with a negative family history (families III and V). Synkinesia has been reported to be associated only with the X-linked form of KS [ 16 ]. However, a recent report identifying the specific gene mutated in autosomal dominant KS pointed out that synkinesia may occur in the autosomal forms of KS [ 17 ]. In families III and V in this report, pedigree analysis strongly pointed to the X-linked form of KS although autosomal recessive inheritance cannot be definitely excluded. Two families were designated as having the autosomal recessive mode of inheritance. One family included affected brother and sister with normal consanguineous parents (family IX), while the other family had 3 affected sisters with normal consanguineous parents (family X). Consanguinity rate among parents of all patients was 83%, with 50% of all marriages being between first cousins.Discussion This study points to the higher proportion of the X-linked form of Kallmann syndrome among all KS cases seen at an endocrine/genetic clinic in Jordan over a period of 5 years. Among 7 families with inherited KS, the X-linked form was the mode of inheritance in 5 families (71% of familial KS). None of the pedigrees was consistent with autosomal dominant inheritance in this series. In the two families with autosomal recessive inheritance, the probability of a non-penetrant autosomal dominant gene in either parent was considered remote because of absence of any relevant family history. Autosomal dominant inheritance was also considered a very remote possibility in the X-linked families because of absence of affected individuals in earlier generations and the pattern of inheritance as revealed by pedigree construction (fig  1 ) was compatible with X-linked inheritance. The X-linked form of KS has at times been reported to account for only one third of inherited cases [ 1 ], and at other times to be the most frequent form [ 2 ]. The high proportion of the X-linked form among our cases may represent a high prevalence of Kal1 gene among Jordanians and Palestinians. Consanguineous marriages in Jordan are favored culturally. Among two thousand marriages in the general population, 32% have been reported to be between first cousins [ 18 ]. The figure of 50% first cousin marriages among parents of our patients would thus reflect the high consanguinity rate among the population in general. Among the XR form of KS, 75% of patients were reported to show synkinesia [ 9 ]. Synkinesia in X-linked KS has been attributed to an abnormal projection of the corticospinal tract [ 19 ]. In our experience, synkinesia was present in 16/22 (73%) patients with the XR form of KS and was characteristically more pronounced in the younger age group. Intrafamilial clinical heterogeneity has been reported among family members carrying the same mutation in Kal 1 gene [ 9 ]. In this series, intrafamilial variability in renal anomalies was exemplified in family I, the largest family in our series, where eight out of eleven patients had renal abnormalities. Intrafamilial variability was also seen in family III, in which one patient had a malrotated kidney, his brother had a horseshoe kidney while the third brother had no renal anomalies. Sensorineural hearing loss has also been reported to be associated mainly with the X-linked form of KS [ 20 ]. The KAL1 gene is expressed in the inner ear from early developmental stages suggesting that the defect underlying the hearing loss in X-linked Kallmann syndrome occurs during the organogenesis period [ 9 ]. In our study, sensorineural hearing impairment was only diagnosed among patients with the X linked form of Kallmann syndrome. Four of nineteen tested males (21%) showed sensorineural hearing impairment with evident intrafamilial variability. In this series cryptorchidism or a history of cryptorchidism was present in 73% of patients (19/26), and was not related to a specific mode of inheritance or etiology. Microphallus was present among 17/26 (65%) patients in this study, with several other patients reporting a history of treatment with testosterone; the exact number of treated patients or the treatment profile could not be precisely determined. Seminal fluid analysis was done for all patients of 16 years and above. The test showed azoospermia for all tested males except patient 1 in family IX, where oligospermia was reported with a sperm count of 10,000,000 per ml. None of the patients with the X-linked form manifested ichthyosis, mental retardation, short stature or ocular albinism, pointing to the underlying etiology being a mutation in the Kal 1 gene rather than a contiguous-gene deletion syndrome [ 21 ]. Olfactory MRI revealed olfactory tract agenesis in 80% of cases for which the investigation was done in the series (19/24). Quinton et al, 1996, indicate that KS may be present with no pathology detected in the olfactory tract on MRI, and that phenotypic characterization of KS was effectively achieved by accurate estimation of olfactory sensation [ 22 ]. Kallmann syndrome has a favorable prognosis under proper management. Its investigation should thus be considered in any child presenting with cryptorchidism and microphallus. Since the gonad state is still dormant in childhood, gonadotrophin levels are not helpful. Olfactory MRI may be a more useful tool for the diagnosis [ 23 ]. Nevertheless, a normal MRI does not rule out Kallmann syndrome as normal olfactory bulbs can be present in up to 25% of cases [ 22 ]. The presence of anosmia/hyposmia and history of delayed puberty or infertility in the family are helpful in establishing the diagnosis. Where diagnosis remains difficult, it is indicated to follow up these children till they reach puberty. The majority of Kallmann syndrome cases in our study showed the X-linked mode of inheritance, which might indicate a high prevalence of Kal1 gene in the population. However, molecular studies for the Kal1 gene were not performed in this study. Patients in our series manifested a wide range of phenotypic heterogeneity with intrafamilial variability of clinical manifestations. We recommend an evaluation for Kallmann syndrome in our population in any child presenting with microphallus and cryptorchidism. Further studies are needed to establish the prevalence rate of Kallmann syndrome in Jordan and to define the causative mutations.
Discussion
This study points to the higher proportion of the X-linked form of Kallmann syndrome among all KS cases seen at an endocrine/genetic clinic in Jordan over a period of 5 years. Among 7 families with inherited KS, the X-linked form was the mode of inheritance in 5 families (71% of familial KS). None of the pedigrees was consistent with autosomal dominant inheritance in this series. In the two families with autosomal recessive inheritance, the probability of a non-penetrant autosomal dominant gene in either parent was considered remote because of absence of any relevant family history. Autosomal dominant inheritance was also considered a very remote possibility in the X-linked families because of absence of affected individuals in earlier generations and the pattern of inheritance as revealed by pedigree construction (fig  1 ) was compatible with X-linked inheritance. The X-linked form of KS has at times been reported to account for only one third of inherited cases [ 1 ], and at other times to be the most frequent form [ 2 ]. The high proportion of the X-linked form among our cases may represent a high prevalence of Kal1 gene among Jordanians and Palestinians. Consanguineous marriages in Jordan are favored culturally. Among two thousand marriages in the general population, 32% have been reported to be between first cousins [ 18 ]. The figure of 50% first cousin marriages among parents of our patients would thus reflect the high consanguinity rate among the population in general.Among the XR form of KS, 75% of patients were reported to show synkinesia [ 9 ]. Synkinesia in X-linked KS has been attributed to an abnormal projection of the corticospinal tract [ 19 ]. In our experience, synkinesia was present in 16/22 (73%) patients with the XR form of KS and was characteristically more pronounced in the younger age group. Intrafamilial clinical heterogeneity has been reported among family members carrying the same mutation in Kal 1 gene [ 9 ]. In this series, intrafamilial variability in renal anomalies was exemplified in family I, the largest family in our series, where eight out of eleven patients had renal abnormalities. Intrafamilial variability was also seen in family III, in which one patient had a malrotated kidney, his brother had a horseshoe kidney while the third brother had no renal anomalies.Sensorineural hearing loss has also been reported to be associated mainly with the X-linked form of KS [ 20 ]. The KAL1 gene is expressed in the inner ear from early developmental stages suggesting that the defect underlying the hearing loss in X-linked Kallmann syndrome occurs during the organogenesis period [ 9 ]. In our study, sensorineural hearing impairment was only diagnosed among patients with the X linked form of Kallmann syndrome. Four of nineteen tested males (21%) showed sensorineural hearing impairment with evident intrafamilial variability.In this series cryptorchidism or a history of cryptorchidism was present in 73% of patients (19/26), and was not related to a specific mode of inheritance or etiology. Microphallus was present among 17/26 (65%) patients in this study, with several other patients reporting a history of treatment with testosterone; the exact number of treated patients or the treatment profile could not be precisely determined. Seminal fluid analysis was done for all patients of 16 years and above. The test showed azoospermia for all tested males except patient 1 in family IX, where oligospermia was reported with a sperm count of 10,000,000 per ml. None of the patients with the X-linked form manifested ichthyosis, mental retardation, short stature or ocular albinism, pointing to the underlying etiology being a mutation in the Kal 1 gene rather than a contiguous-gene deletion syndrome [ 21 ].Olfactory MRI revealed olfactory tract agenesis in 80% of cases for which the investigation was done in the series (19/24). Quinton et al, 1996, indicate that KS may be present with no pathology detected in the olfactory tract on MRI, and that phenotypic characterization of KS was effectively achieved by accurate estimation of olfactory sensation [ 22 ]. Kallmann syndrome has a favorable prognosis under proper management. Its investigation should thus be considered in any child presenting with cryptorchidism and microphallus. Since the gonad state is still dormant in childhood, gonadotrophin levels are not helpful. Olfactory MRI may be a more useful tool for the diagnosis [ 23 ]. Nevertheless, a normal MRI does not rule out Kallmann syndrome as normal olfactory bulbs can be present in up to 25% of cases [ 22 ]. The presence of anosmia/hyposmia and history of delayed puberty or infertility in the family are helpful in establishing the diagnosis. Where diagnosis remains difficult, it is indicated to follow up these children till they reach puberty.The majority of Kallmann syndrome cases in our study showed the X-linked mode of inheritance, which might indicate a high prevalence of Kal1 gene in the population. However, molecular studies for the Kal1 gene were not performed in this study. Patients in our series manifested a wide range of phenotypic heterogeneity with intrafamilial variability of clinical manifestations. We recommend an evaluation for Kallmann syndrome in our population in any child presenting with microphallus and cryptorchidism. Further studies are needed to establish the prevalence rate of Kallmann syndrome in Jordan and to define the causative mutations.Competing interests The authors declare that they have no competing interests.
Competing interests
The authors declare that they have no competing interests.Authors' contributions MAJ and KA were the main researchers. HH and MAJ drafted the manuscript. All authors were part of the team that evaluated the patients
Authors' contributions
MAJ and KA were the main researchers. HH and MAJ drafted the manuscript. All authors were part of the team that evaluated the patients


The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif in Rpn11 and Csn5 underlies isopeptidase activities intrinsic to the proteasome and signalosome, respectively. We show here that the archaebacterial protein AfJAMM possesses the key features of a zinc metalloprotease, yet with a distinct fold. The histidine and aspartic acid of the conserved EX n HS/THX 7 SXXD motif coordinate a zinc, whereas the glutamic acid hydrogen-bonds an aqua ligand. By analogy to the active site of thermolysin, we predict that the glutamic acid serves as an acid-base catalyst and the second serine stabilizes a tetrahedral intermediate. Mutagenesis of Csn5 confirms these residues are required for Nedd8 isopeptidase activity. The active site-like architecture specified by the JAMM motif motivates structure-based approaches to the study of JAMM domain proteins and the development of therapeutic proteasome and signalosome inhibitors.

Introduction Many cellular proteins are degraded by the proteasome after they become covalently modified with a multiubiquitin chain. The 26S proteasome is a massive protein composed of a 20S core and two 19S regulatory particles ( Voges et al. 1999 ). The 20S core can be subdivided into a dimer of heptameric rings of β subunits—which contain the proteolytic active sites responsible for the protein degradation activity of the proteasome—flanked by heptameric rings of α subunits. The 19S regulatory particle can be divided into a base thought to comprise a hexameric ring of AAA ATPases and a lid composed of eight or more distinct subunits. Whereas 20S core particles and AAA ATPase rings have been found in compartmentalized proteases in prokaryotes, the lid domain of the 19S regulatory particle is unique to eukaryotes and provides the specificity of 26S proteasomes for ubiquitinated substrates ( Glickman et al. 1998 ). Ubiquitin (Ub), an 8 kD protein, is conjugated by Ub ligases to proteasome substrates via an isopeptide bond that links its carboxyl terminus to the amino sidechain of a lysine residue in the substrate. Ub-like proteins (Ubls), of which there are several, are conjugated to their target proteins in a similar manner. Ubls typically do not promote degradation of their targets by the proteasome, but rather regulate target activity in a more subtle manner reminiscent of protein phosphorylation ( Hershko and Ciechanover 1998 ;  Peters et al. 1998 ). As is the case for protein phosphorylation, the attachment of Ub and Ubls to target proteins is opposed by isopeptidase enzymes that undo the handiwork of Ub ligases. For example, removal of the Ubl Nedd8 (neural precursor cell expressed, developmentally downregulated 8) regulatory modification from the Cullin 1 (Cul1) subunit of the SCF (Skp1/Cdc53/Cullin/F-box receptor) Ub ligase is catalyzed by the COP9 signalosome (CSN) ( Lyapina et al. 2001 ). The CSN was identified in  Arabidopsis thaliana  from genetic studies of constitutively photomorphogenic mutant plants ( Osterlund et al. 1999 ). It later became evident that CSN and the proteasome lid are paralogous complexes ( Glickman et al. 1998 ;  Seeger et al. 1998 ;  Wei et al. 1998 ). Csn5 of CSN and Rpn11 (regulatory particle number 11) of the proteasome lid are the subunits that are most closely related between the two complexes. CSN-dependent isopeptidase activity is sensitive to metal ion chelators, and Csn5 contains a conserved, putative metal-binding motif (EX n HS/THX 7 SXXD), referred to as the JAMM motif, that is embedded within the larger JAB1/MPN/Mov34 domain (hereafter referred to as the JAMM domain) and is critical for Csn5 function in vivo ( Cope et al. 2002 ). Removal of Ub from proteasome substrates is also promoted by a metal ion-dependent isopeptidase activity associated with the proteasome ( Verma et al. 2002 ;  Yao and Cohen 2002 ). The JAMM/MPN +  motif of Rpn11 is critical for its function in vivo ( Maytal-Kivity et al. 2002 ;  Verma et al. 2002 ;  Yao and Cohen 2002 ), and proteasomes that contain Rpn11 bearing a mutated JAMM motif are unable to promote deubiquitination and degradation of the proteasome substrate Sic1 ( Verma et al. 2002 ). Taken together, these observations suggested that the JAMM motif specifies a catalytic center that in turn defines a novel family of metalloisopeptidases. Interestingly, the JAMM motif is found in proteins from all three domains of life ( Cope et al. 2002 ;  Maytal-Kivity et al. 2002 ), indicating that it has functions beyond the Ub system. In this study, we present the crystal structure of the  Archaeoglobus fulgidus AF2198  gene product AfJAMM and explore the implications of its novel metalloprotease architecture.
Introduction
Many cellular proteins are degraded by the proteasome after they become covalently modified with a multiubiquitin chain. The 26S proteasome is a massive protein composed of a 20S core and two 19S regulatory particles ( Voges et al. 1999 ). The 20S core can be subdivided into a dimer of heptameric rings of β subunits—which contain the proteolytic active sites responsible for the protein degradation activity of the proteasome—flanked by heptameric rings of α subunits. The 19S regulatory particle can be divided into a base thought to comprise a hexameric ring of AAA ATPases and a lid composed of eight or more distinct subunits. Whereas 20S core particles and AAA ATPase rings have been found in compartmentalized proteases in prokaryotes, the lid domain of the 19S regulatory particle is unique to eukaryotes and provides the specificity of 26S proteasomes for ubiquitinated substrates ( Glickman et al. 1998 ). Ubiquitin (Ub), an 8 kD protein, is conjugated by Ub ligases to proteasome substrates via an isopeptide bond that links its carboxyl terminus to the amino sidechain of a lysine residue in the substrate. Ub-like proteins (Ubls), of which there are several, are conjugated to their target proteins in a similar manner. Ubls typically do not promote degradation of their targets by the proteasome, but rather regulate target activity in a more subtle manner reminiscent of protein phosphorylation ( Hershko and Ciechanover 1998 ;  Peters et al. 1998 ).As is the case for protein phosphorylation, the attachment of Ub and Ubls to target proteins is opposed by isopeptidase enzymes that undo the handiwork of Ub ligases. For example, removal of the Ubl Nedd8 (neural precursor cell expressed, developmentally downregulated 8) regulatory modification from the Cullin 1 (Cul1) subunit of the SCF (Skp1/Cdc53/Cullin/F-box receptor) Ub ligase is catalyzed by the COP9 signalosome (CSN) ( Lyapina et al. 2001 ). The CSN was identified in  Arabidopsis thaliana  from genetic studies of constitutively photomorphogenic mutant plants ( Osterlund et al. 1999 ). It later became evident that CSN and the proteasome lid are paralogous complexes ( Glickman et al. 1998 ;  Seeger et al. 1998 ;  Wei et al. 1998 ). Csn5 of CSN and Rpn11 (regulatory particle number 11) of the proteasome lid are the subunits that are most closely related between the two complexes. CSN-dependent isopeptidase activity is sensitive to metal ion chelators, and Csn5 contains a conserved, putative metal-binding motif (EX n HS/THX 7 SXXD), referred to as the JAMM motif, that is embedded within the larger JAB1/MPN/Mov34 domain (hereafter referred to as the JAMM domain) and is critical for Csn5 function in vivo ( Cope et al. 2002 ). Removal of Ub from proteasome substrates is also promoted by a metal ion-dependent isopeptidase activity associated with the proteasome ( Verma et al. 2002 ;  Yao and Cohen 2002 ). The JAMM/MPN +  motif of Rpn11 is critical for its function in vivo ( Maytal-Kivity et al. 2002 ;  Verma et al. 2002 ;  Yao and Cohen 2002 ), and proteasomes that contain Rpn11 bearing a mutated JAMM motif are unable to promote deubiquitination and degradation of the proteasome substrate Sic1 ( Verma et al. 2002 ). Taken together, these observations suggested that the JAMM motif specifies a catalytic center that in turn defines a novel family of metalloisopeptidases. Interestingly, the JAMM motif is found in proteins from all three domains of life ( Cope et al. 2002 ;  Maytal-Kivity et al. 2002 ), indicating that it has functions beyond the Ub system. In this study, we present the crystal structure of the  Archaeoglobus fulgidus AF2198  gene product AfJAMM and explore the implications of its novel metalloprotease architecture.Archaeoglobus fulgidus AF2198Results and Discussion We proposed that the subset of JAMM domain proteins that contain a JAMM motif comprise a novel family of metallopeptidases ( Cope et al. 2002 ). To gain a clearer understanding of these putative enzymes—in particular the pertinent subunits of the proteasome lid and signalosome ( Figure 1 )—we cloned and expressed in  Escherichia coli  a variety of JAMM motif-containing proteins to find a suitable candidate for crystallographic analysis. The expression of all candidates except for AfJAMM led to insoluble aggregates. Unlike many JAMM proteins that contain an additional domain, the AfJAMM protein consists entirely of the JAMM domain. We were able to purify and crystallize native and selenomethionine-substituted AfJAMM; the latter was used for phasing by employing the multiwavelength anomalous diffraction (MAD) technique (see  Table 1  for statistics). Figure 1 Alignment of Eukaryotic JAMM Domains with AfJAMM Eukaryotic JAMM domain proteins were aligned with AfJAMM using ClustalX and manually refined. Sequences are named with a two-letter code corresponding to the genus and species of the respective organism followed by the name of the protein (see Supporting Information for accession numbers), and ‘hyp’ is an abbreviation for hypothetical. The JAMM motif comprises the residues highlighted in green (E22, H67, H69, S77, and D80), and the active site core is surrounded by a red box. Conserved residues are highlighted in gray. The disulfide cysteine residues are highlighted in yellow (C74, C95). Active site residues that were mutated in  S. pombe  Csn5 are marked with an asterisk beneath the alignment. The secondary structure of AfJAMM is indicated above the sequence; helices are blue, sheets are red arrows, and loops are yellow lines. The dashed yellow line indicates a loop (F42–G58) that is disordered in the crystal. Table 1 Data Collection Statistics
 a Owing to pseudocentering, reflections with l values such that cos 2  (0.54πl) < ½ are systematically weak, leading to an R-factor higher than would be expected for a nonpseudocentered crystal structure. R N  are the R-factors calculated with only the reflections with cos 2  (0.54πl) > ½ (see Materials and Methods). Barring rearrangements of sidechains in the vicinity of the zinc atom, no significant changes were seen between the native and selenomethionine forms AfJAMM consists of an eight-stranded β sheet (β1–β8), flanked by a long α helix (α1) between the first and second strand, and a short α helix (α2) between the fourth and fifth strand. This β sheet resembles a β barrel halved longitudinally and curled around α1 ( Figure 2 A). The α2 helix is oriented lengthwise on the convex surface of the β sheet. The zinc-binding site is adjacent to a loop that spans the end of β4 to the beginning of α2 and is stabilized by a disulfide bond between C74 from this loop to C95 on β5. Although disulfide bonds are scarce in intracellular proteins, they are often present in homologous proteins found in hyperthermophiles ( Mallick et al. 2002 ). The overall fold resembles that of the zinc metalloenzyme cytidine deaminase (CDA). CDA from  Bacillus subtilis  ( Johansson et al. 2002 ) can be superimposed onto AfJAMM with a root-mean squared (RMS) deviation of 3.0 Å over 79 α carbons, despite only 9% sequence identity over structurally aligned residues. The catalytic zinc ions of AfJAMM and CDA, 4.9 Å apart in the superposition, occupy the same general vicinity in the tertiary structures but are coordinated by entirely different protein ligands, two histidines and an aspartic acid in AfJAMM compared to three cysteines in CDA, located at different positions in the sequence ( Figure 2 A). Consequently, the JAMM fold represents a departure from the papain-like cysteine protease architecture that underlies the deubiquitinating activity of the most thoroughly characterized deubiquitinating enzymes (DUBs), the Ub carboxy-terminal hydrolases (UCHs) ( Johnston et al. 1997 ) and Ub-specific proteases (UBPs) ( Hu et al. 2002 ). Figure 2 Crystal Structure of AfJAMM (A) On the left, the AfJAMM protomer is presented. The amino and carboxyl termini are marked by N and C. The catalytic zinc atom is depicted as a gray sphere. The zinc ligands (H67, H69, and D80) are colored in green. Secondary structure elements are numbered α1–α2 and β1–β8. The amino acids that mark the beginning and end of the disordered loop (P41–M60) are labeled. On the right, the crystal structure of the cytidine deaminase protomer is shown in the same orientation as AfJAMM to highlight the fold likeness as well as the similarly situated zinc-binding sites. The zinc ligands (C53, C86, and C89) are colored in green. (B) The dimer in the asymmetric unit of AfJAMM crystals. The side view is obtained by rotating the monomer in (A) by 90° as indicated by the quarter-arrow around the y-axis. The gold protomer is related to the green protomer by a 180° rotation around the crystallographic c-axis (shown as a black bar in the side view) and a translation of 3.38 Å. The two AfJAMM subunits in the asymmetric unit are connected through a parallel β sheet formed at the dimer interface ( Figure 2 B). The subunits are related by a 2-fold screw axis along the crystallographic c-axis with a translation of 3.38 Å, corresponding to a displacement of one residue along the β3 strand. AfJAMM behaves as a monomer during size exclusion chromatography, suggesting that the dimer observed in the asymmetric unit is an artifact of crystallization. Yet the residues of β3 are highly conserved among JAMM proteins (see  Figure 1 ) and predominantly hydrophobic, which makes it difficult to regard the observed interaction as completely insignificant. Flanking β3 to the carboxy-terminal side, there is a striking covariation of residues, MPQSGTG in Rpn11 orthologues and LPVEGTE in Csn5 orthologues. The potential of β3 and the flanking region to mediate specific protein–protein interactions, such as the assembly of Rpn11 and Csn5 into their respective complexes or their specificity towards Ub or Nedd8, warrants further investigation. The zinc-binding site of AfJAMM is located in a furrow formed by the convex surface of the β2–β4 sheet and α2. The catalytic zinc has a tetrahedral coordination sphere ( Figure 3 A), with ligands provided by N ɛ2  of H67 and H69 on β4, the carboxylate of D80 on α2, and a water molecule. The latter hydrogen-bonds to the sidechain of E22 on β2. Thus, the crystal structure confirms previous predictions that the histidine and aspartic acid residues in the JAMM motif are ligands for a metal ( Cope et al. 2002 ;  Verma et al. 2002 ;  Yao and Cohen 2002 ). It must be noted that the identity of the physiological metal in AfJAMM and eukaryotic JAMM homologues is still unknown. The majority of metalloproteases naturally employ zinc but show altered activities with other substituted metals ( Auld 1995 ). Figure 3 Metalloprotease-Like Active Site of AfJAMM (A) The active site of AfJAMM is shown centered around the catalytic zinc ion, which is represented as a dark gray sphere surrounded by anomalous cross Fourier difference density (contoured at 9.5 σ) colored in red. The aqua ligand, which lies at 2.9 Å from the zinc, is shown as a red sphere surrounded by purple density (contoured at 3 σ) of an F obs  – F calc  map, in which the aqua ligand was omitted from the calculation. Residues that underlie isopeptide bond cleavage are shown in green. The carboxylate oxygen atoms of D80 lie 2.2 Å from the zinc. The N ɛ2  atoms of H67 and H69 lie 2.1 Å from the zinc. The carboxylate oxygen atoms of E22 lie 3.2–3.5 Å from the aqua ligand and 4.5–5.0 Å from the zinc. Ancillary active site residues and the backbone (ribbon diagram) are shown in grey. The disulfide bond that links C74 to C95 is shown in yellow. The JAMM motif is shown in the upper lefthand corner for reference. (B) Superimposition of active site residues in ScNP, thermolysin, and AfJAMM. AfJAMM is in green, ScNP in blue, and thermolysin in red. For clarity only, the sidechains from the residues that bind the zinc or aqua ligands are shown in their entirety. In addition, atoms that stabilize the putative tetrahedral intermediate are shown. These include O γ  of S77 in AfJAMM, O η  of Y95 in ScNP, and the N ɛ2  of H231 in thermolysin. The arrangement of zinc ligands in AfJAMM resembles that found in thermolysin, the  Streptomyces caespitosus  zinc endoprotease (ScNP), and neurolysin, a mammalian metalloprotease ( Kurisu et al. 2000 ;  Brown et al. 2001 ;  English et al. 2001 ). Thermolysin, neurolysin, and ScNP are homologues that have the classical HEXXH metalloprotease motif and adopt the same core fold. In contrast, the sequence, zinc-binding motif, and fold adopted by AfJAMM are entirely distinct. Nonetheless, the active site metal and ligand atoms of thermolysin and ScNP can be superimposed on those of AfJAMM with an RMS deviation of approximately 0.4–0.5 Å ( Figure 3 B). While this manuscript was under revision, an independent report of a crystal structure of the  AF2198  gene product appeared ( Tran et al. 2003 ). These authors used the fold similarity to CDA as a framework to evaluate the function of the JAMM motif. Given the biochemical data supporting the JAMM motif's role in proteolysis, the common active site architecture seen in AfJAMM and thermolysin, and the similarity of zinc ligands between thermolysin and AfJAMM, we believe that the extensive body of mechanistic studies on thermolysin and related metalloproteases provide a better framework for the analysis of JAMM function than CDA. In addition to the correspondence between zinc ligands, the glutamic acid residue (E166) downstream of the HEXXH motif of thermolysin is functionally equivalent to the aspartic acid ligand of AfJAMM (D80). E22 in AfJAMM is functionally equivalent to the glutamic acid in thermolysin's HEXXH motif, which serves as the general acid-base catalyst. The conserved serine between the histidine ligands interacts with E22 through a sidechain–main chain hydrogen bond. In more distant JAMM relatives, the serine is replaced by a threonine or asparagine ( Aravind and Ponting 1998 ), both of which are capable of the same bracing function. Meanwhile, the γ-hydroxyl group of the highly conserved S77 in AfJAMM occupies a position similar to N ɛ2  of H231 in thermolysin. This atom flanks the ‘oxyanion hole’ and is implicated in stabilizing the tetrahedral intermediate formed during hydrolysis of the scissile bond ( Matthews 1988 ;  Lipscomb and Strater 1996 ). AfJAMM was tested for the ability to hydrolyze a number of substrates, including Ub derivatives, resofurin-labeled casein, and D-alanine compounds. Unfortunately, none of the in vitro assays yielded positive results. As nothing is known about AfJAMM in the context of  A. fulgidus  biology, these negative results do not rule out the possibility that AfJAMM functions as a peptide hydrolase in vivo. To validate the suitability of the AfJAMM structure as a basic model for eukaryotic JAMM proteins, we performed site-directed mutagenesis of  Schizosaccharomyces pombe csn5 + . The zinc ligands of Csn5 were previously established to be essential for its role in sustaining cleavage of the isopeptide bond that links Nedd8 to Cul1 ( Cope et al. 2002 ). Alanine substitutions for the putative general acid-base catalyst (E56A) and the catalytic serine (S128) in the JAMM motif of Csn5 likewise abolished its ability to remove the Nedd8 moiety from Cul1 in a  csn5
 +  background ( Figure 4 A). The E56A mutation had no effect on the assembly of Csn5 with Csn1 myc13 , while assembly with S128A was slightly hindered ( Figure 4 A). Mutation of the equivalent serine codon in  RPN11  destroyed complementing activity without altering assembly of Rpn11 into the lid. However, the effect of this mutation on Rpn11 isopeptidase activity was not evaluated ( Maytal-Kivity et al. 2002 ). Alanine substitutions for a catalytic residue (E56) or zinc ligands (H118A, D131N) exerted a modest dominant-negative phenotype in  csn5 +  cells ( Figure 4 B). Figure 4 Mutations in the JAMM Motif of Csn5 Abrogate the Deneddylating Activity of the CSN (A) Mutations in the glutamic acid (E56A) that positions the aqua ligand and in the proposed catalytic serine (S128A) of Csn5 disrupt deneddylation of Cul1 by CSN but have no effect on assembly with Csn1. A  csn5Δ  strain of  S. pombe  was transformed with an empty pREP-41 plasmid (lane 1) or with the plasmid encoding FLAG tagged: Csn5 (lane 2), Csn5 E56A  (lane 3), or Csn5 S128A  (lane 4). Whole-cell lysates were used for Western blot analysis with anti-Cul1 antibodies (top gel) and anti-FLAG antibodies (second from top). A strain with a  myc13 -tagged Csn1 was transformed with the above plasmids, and whole-cell lysates were used for Western blot analysis. Antibodies to the Myc tag were used to detect Csn1 myc13  (third from top), and were used to pull down Csn1 myc13  and subsequently blot with anti-FLAG antibodies to detect coprecipitated Csn5 mutant proteins (bottom gel). (B) Mutations in the JAMM motif display a modest dominant-negative phenotype. Western blot analysis of crude cell lysates was performed as described in (A). (C) Selected JAMM motifs from proteins of diverse functions. The canonical JAMM motif residues are highlighted in green. The conserved proline is highlighted in blue, and semiconserved cysteine is highlighted in yellow. We have been able to assign biochemical functions to Csn5 and Rpn11 ( Cope et al. 2002 ;  Verma et al. 2002 ;  Yao and Cohen 2002 ), but the functions of other eukaryotic JAMM proteins ( Figure 4 C) such as AMSH and C6.1A, as well as the prokaryotic protein RadC and the viral phage λ tail assembly protein K, remain unknown. The structure of AfJAMM provides a useful tool for dissecting the functions of JAMM motifs in these varied contexts and inspires the search for specific JAMM active site inhibitors. The mechanistic implications of the AfJAMM structure explain why the deubiquitinating activity of the lid was unaffected by inhibitors of classical DUBs, the UCHs and UBPs. In classical DUBs, the nucleophile that attacks the carbon of the scissile bond is provided by a cysteine residue in the active site. This property is exploited by using the irreversible inhibitor Ub–aldehyde, which forms a nonhydrolyzable bond to the nucleophilic cysteine ( Johnston et al. 1999 ). In contrast, JAMM proteins likely hydrolyze Ub conjugates in a manner similar to thermolysin, in which the zinc-polarized aqua ligand serves as the nucleophile ( Lipscomb and Strater 1996 ). In the case of thermolysin, metal chelators and phosphonamidate peptides are effective inhibitors ( Bartlett and Marlowe 1987 ), whereas other zinc metalloproteases are sensitive to peptidomimetic substrates bearing a hydroxamate group ( Skiles et al. 2001 ). Metal chelators have been shown to be effective inhibitors of JAMM proteins ( Cope et al. 2002 ;  Verma et al. 2002 ); it would be interesting to see whether phosphonamidate and hydroxamate peptide mimics of Ub conjugate isopeptides would be equally effective. The proteasome inhibitor PS-341 has gained attention for its novelty and effectiveness in treating various forms of cancer ( Adams 2002 ). PS-341 was recently approved by the United States' Food and Drug Administration for treatment of relapsed multiple myeloma, thereby validating the proteasome as a target for anticancer therapies. The active site of JAMM proteins is an intriguing target for second-generation therapeutics targeted at the Ub–proteasome pathway for two reasons: the JAMM motif in the proteasome lid is essential for the proteasome to function and the JAMM motif in the CSN specifically regulates the activity of a critical family of E3 Ub ligases ( Nalepa and Harper 2003 ). Inhibition of SCF and other Cullin-based ligases by way of the JAMM motif may be a more specific means of modulating levels of key proteasome substrates in cancer cells.
Results and Discussion
We proposed that the subset of JAMM domain proteins that contain a JAMM motif comprise a novel family of metallopeptidases ( Cope et al. 2002 ). To gain a clearer understanding of these putative enzymes—in particular the pertinent subunits of the proteasome lid and signalosome ( Figure 1 )—we cloned and expressed in  Escherichia coli  a variety of JAMM motif-containing proteins to find a suitable candidate for crystallographic analysis. The expression of all candidates except for AfJAMM led to insoluble aggregates. Unlike many JAMM proteins that contain an additional domain, the AfJAMM protein consists entirely of the JAMM domain. We were able to purify and crystallize native and selenomethionine-substituted AfJAMM; the latter was used for phasing by employing the multiwavelength anomalous diffraction (MAD) technique (see  Table 1  for statistics).Data Collection Statistics
a Owing to pseudocentering, reflections with l values such that cos 2  (0.54πl) < ½ are systematically weak, leading to an R-factor higher than would be expected for a nonpseudocentered crystal structure. R N  are the R-factors calculated with only the reflections with cos 2  (0.54πl) > ½ (see Materials and Methods). Barring rearrangements of sidechains in the vicinity of the zinc atom, no significant changes were seen between the native and selenomethionine formsAfJAMM consists of an eight-stranded β sheet (β1–β8), flanked by a long α helix (α1) between the first and second strand, and a short α helix (α2) between the fourth and fifth strand. This β sheet resembles a β barrel halved longitudinally and curled around α1 ( Figure 2 A). The α2 helix is oriented lengthwise on the convex surface of the β sheet. The zinc-binding site is adjacent to a loop that spans the end of β4 to the beginning of α2 and is stabilized by a disulfide bond between C74 from this loop to C95 on β5. Although disulfide bonds are scarce in intracellular proteins, they are often present in homologous proteins found in hyperthermophiles ( Mallick et al. 2002 ). The overall fold resembles that of the zinc metalloenzyme cytidine deaminase (CDA). CDA from  Bacillus subtilis  ( Johansson et al. 2002 ) can be superimposed onto AfJAMM with a root-mean squared (RMS) deviation of 3.0 Å over 79 α carbons, despite only 9% sequence identity over structurally aligned residues. The catalytic zinc ions of AfJAMM and CDA, 4.9 Å apart in the superposition, occupy the same general vicinity in the tertiary structures but are coordinated by entirely different protein ligands, two histidines and an aspartic acid in AfJAMM compared to three cysteines in CDA, located at different positions in the sequence ( Figure 2 A). Consequently, the JAMM fold represents a departure from the papain-like cysteine protease architecture that underlies the deubiquitinating activity of the most thoroughly characterized deubiquitinating enzymes (DUBs), the Ub carboxy-terminal hydrolases (UCHs) ( Johnston et al. 1997 ) and Ub-specific proteases (UBPs) ( Hu et al. 2002 ).The two AfJAMM subunits in the asymmetric unit are connected through a parallel β sheet formed at the dimer interface ( Figure 2 B). The subunits are related by a 2-fold screw axis along the crystallographic c-axis with a translation of 3.38 Å, corresponding to a displacement of one residue along the β3 strand. AfJAMM behaves as a monomer during size exclusion chromatography, suggesting that the dimer observed in the asymmetric unit is an artifact of crystallization. Yet the residues of β3 are highly conserved among JAMM proteins (see  Figure 1 ) and predominantly hydrophobic, which makes it difficult to regard the observed interaction as completely insignificant. Flanking β3 to the carboxy-terminal side, there is a striking covariation of residues, MPQSGTG in Rpn11 orthologues and LPVEGTE in Csn5 orthologues. The potential of β3 and the flanking region to mediate specific protein–protein interactions, such as the assembly of Rpn11 and Csn5 into their respective complexes or their specificity towards Ub or Nedd8, warrants further investigation.The zinc-binding site of AfJAMM is located in a furrow formed by the convex surface of the β2–β4 sheet and α2. The catalytic zinc has a tetrahedral coordination sphere ( Figure 3 A), with ligands provided by N ɛ2  of H67 and H69 on β4, the carboxylate of D80 on α2, and a water molecule. The latter hydrogen-bonds to the sidechain of E22 on β2. Thus, the crystal structure confirms previous predictions that the histidine and aspartic acid residues in the JAMM motif are ligands for a metal ( Cope et al. 2002 ;  Verma et al. 2002 ;  Yao and Cohen 2002 ). It must be noted that the identity of the physiological metal in AfJAMM and eukaryotic JAMM homologues is still unknown. The majority of metalloproteases naturally employ zinc but show altered activities with other substituted metals ( Auld 1995 ).The arrangement of zinc ligands in AfJAMM resembles that found in thermolysin, the  Streptomyces caespitosus  zinc endoprotease (ScNP), and neurolysin, a mammalian metalloprotease ( Kurisu et al. 2000 ;  Brown et al. 2001 ;  English et al. 2001 ). Thermolysin, neurolysin, and ScNP are homologues that have the classical HEXXH metalloprotease motif and adopt the same core fold. In contrast, the sequence, zinc-binding motif, and fold adopted by AfJAMM are entirely distinct. Nonetheless, the active site metal and ligand atoms of thermolysin and ScNP can be superimposed on those of AfJAMM with an RMS deviation of approximately 0.4–0.5 Å ( Figure 3 B).While this manuscript was under revision, an independent report of a crystal structure of the  AF2198  gene product appeared ( Tran et al. 2003 ). These authors used the fold similarity to CDA as a framework to evaluate the function of the JAMM motif. Given the biochemical data supporting the JAMM motif's role in proteolysis, the common active site architecture seen in AfJAMM and thermolysin, and the similarity of zinc ligands between thermolysin and AfJAMM, we believe that the extensive body of mechanistic studies on thermolysin and related metalloproteases provide a better framework for the analysis of JAMM function than CDA. In addition to the correspondence between zinc ligands, the glutamic acid residue (E166) downstream of the HEXXH motif of thermolysin is functionally equivalent to the aspartic acid ligand of AfJAMM (D80). E22 in AfJAMM is functionally equivalent to the glutamic acid in thermolysin's HEXXH motif, which serves as the general acid-base catalyst. The conserved serine between the histidine ligands interacts with E22 through a sidechain–main chain hydrogen bond. In more distant JAMM relatives, the serine is replaced by a threonine or asparagine ( Aravind and Ponting 1998 ), both of which are capable of the same bracing function. Meanwhile, the γ-hydroxyl group of the highly conserved S77 in AfJAMM occupies a position similar to N ɛ2  of H231 in thermolysin. This atom flanks the ‘oxyanion hole’ and is implicated in stabilizing the tetrahedral intermediate formed during hydrolysis of the scissile bond ( Matthews 1988 ;  Lipscomb and Strater 1996 ).AF2198AfJAMM was tested for the ability to hydrolyze a number of substrates, including Ub derivatives, resofurin-labeled casein, and D-alanine compounds. Unfortunately, none of the in vitro assays yielded positive results. As nothing is known about AfJAMM in the context of  A. fulgidus  biology, these negative results do not rule out the possibility that AfJAMM functions as a peptide hydrolase in vivo. To validate the suitability of the AfJAMM structure as a basic model for eukaryotic JAMM proteins, we performed site-directed mutagenesis of  Schizosaccharomyces pombe csn5 + . The zinc ligands of Csn5 were previously established to be essential for its role in sustaining cleavage of the isopeptide bond that links Nedd8 to Cul1 ( Cope et al. 2002 ). Alanine substitutions for the putative general acid-base catalyst (E56A) and the catalytic serine (S128) in the JAMM motif of Csn5 likewise abolished its ability to remove the Nedd8 moiety from Cul1 in a  csn5
 +  background ( Figure 4 A). The E56A mutation had no effect on the assembly of Csn5 with Csn1 myc13 , while assembly with S128A was slightly hindered ( Figure 4 A). Mutation of the equivalent serine codon in  RPN11  destroyed complementing activity without altering assembly of Rpn11 into the lid. However, the effect of this mutation on Rpn11 isopeptidase activity was not evaluated ( Maytal-Kivity et al. 2002 ). Alanine substitutions for a catalytic residue (E56) or zinc ligands (H118A, D131N) exerted a modest dominant-negative phenotype in  csn5 +  cells ( Figure 4 B).Schizosaccharomyces pombe csn5 +csn5myc13RPN11csn5 +We have been able to assign biochemical functions to Csn5 and Rpn11 ( Cope et al. 2002 ;  Verma et al. 2002 ;  Yao and Cohen 2002 ), but the functions of other eukaryotic JAMM proteins ( Figure 4 C) such as AMSH and C6.1A, as well as the prokaryotic protein RadC and the viral phage λ tail assembly protein K, remain unknown. The structure of AfJAMM provides a useful tool for dissecting the functions of JAMM motifs in these varied contexts and inspires the search for specific JAMM active site inhibitors. The mechanistic implications of the AfJAMM structure explain why the deubiquitinating activity of the lid was unaffected by inhibitors of classical DUBs, the UCHs and UBPs. In classical DUBs, the nucleophile that attacks the carbon of the scissile bond is provided by a cysteine residue in the active site. This property is exploited by using the irreversible inhibitor Ub–aldehyde, which forms a nonhydrolyzable bond to the nucleophilic cysteine ( Johnston et al. 1999 ). In contrast, JAMM proteins likely hydrolyze Ub conjugates in a manner similar to thermolysin, in which the zinc-polarized aqua ligand serves as the nucleophile ( Lipscomb and Strater 1996 ). In the case of thermolysin, metal chelators and phosphonamidate peptides are effective inhibitors ( Bartlett and Marlowe 1987 ), whereas other zinc metalloproteases are sensitive to peptidomimetic substrates bearing a hydroxamate group ( Skiles et al. 2001 ). Metal chelators have been shown to be effective inhibitors of JAMM proteins ( Cope et al. 2002 ;  Verma et al. 2002 ); it would be interesting to see whether phosphonamidate and hydroxamate peptide mimics of Ub conjugate isopeptides would be equally effective.The proteasome inhibitor PS-341 has gained attention for its novelty and effectiveness in treating various forms of cancer ( Adams 2002 ). PS-341 was recently approved by the United States' Food and Drug Administration for treatment of relapsed multiple myeloma, thereby validating the proteasome as a target for anticancer therapies. The active site of JAMM proteins is an intriguing target for second-generation therapeutics targeted at the Ub–proteasome pathway for two reasons: the JAMM motif in the proteasome lid is essential for the proteasome to function and the JAMM motif in the CSN specifically regulates the activity of a critical family of E3 Ub ligases ( Nalepa and Harper 2003 ). Inhibition of SCF and other Cullin-based ligases by way of the JAMM motif may be a more specific means of modulating levels of key proteasome substrates in cancer cells.Materials and Methods The gene for  A. fulgidus  JAMM ( Ponting et al. 1999 ), open reading frame  AF2198 , was cloned from genomic DNA (ATCC #49558D; American Type Culture Collection, Manassas, Virginia, United States) into the pCRT7 vectors (Invitrogen, Carlsbad, California, United States). During cloning, the alternate start codon, GTG, was replaced with the canonical start codon, ATG. The construct was expressed in BL21(DE3)pLysS cells (Novagen, Madison, Wisconsin, United States). The cells were grown to midlog phase in terrific broth media and induced with 0.5 mM IPTG. The cells were lysed by sonication and the protein was isolated by immobilized metal ion chromatography using a Ni-NTA resin (Qiagen, Valencia, California, United States). The protein was further purified by gel filtration on a Sephacryl S100 column (Amersham Pharmacia Biotech, Chalfont St Giles, United Kingdom) and concentrated. The amino-terminal tag was removed by limited digestion with trypsin. Mass spectrometry analysis revealed that trypsin only cut AfJAMM in the amino-terminal tag region, and only a single band was evident on a Coomassie-stained polyacrylamide gel. The tag and uncut protein were removed with Ni-NTA resin followed by anion-exchange chromatography with SOURCE 30Q resin (Amersham Pharmacia Biotech). The processed protein was then concentrated to approximately 30 mg/ml by ultrafiltration. The selenomethionine protein was produced as described elsewhere ( Van Duyne et al. 1993 ) and purified using the same protocol as for the native protein. Protein crystals were obtained in 100 mM NH 4 H 2 PO 4 , 200 mM sodium citrate (pH 5) using vapor diffusion with sitting drops and hanging drops. Crystals were incubated for approximately 1 min in a cryo-solution of equal volumes of reservoir solution and 35% meso-erythritol for the selenomethionine crystals and supplemented with 5 mM ZnCl 2  for the native crystals. The crystals belonged to the space group P6 5 , with cell dimensions of a = b = 76 Å, c = 94 Å and two subunits per asymmetric unit. Data for the selenomethionine crystals were collected on Beamline 9.2 at the Stanford Synchrotron Radiation Laboratory (SSRL) (Stanford, California, United States) and data for the native crystals were collected on Beamline 8.2.1 at the Advanced Light Source (ALS) (Lawrence Berkeley National Laboratory, Berkeley, California, United States) (see  Table 1 ). Phases were obtained by the MAD technique using data collected from selenomethionine-substituted crystals (see  Table 1 ). Three Se atoms were located by SOLVE ( Terwilliger and Berendzen 1999 ) and used to calculate the initial phases. Phasing was subsequently improved by noncrystallographic symmetry averaging, using operators derived from the Se positions, and solvent flattening in RESOLVE ( Terwilliger 2000 ). The polypeptide model was built in O ( Jones et al. 1991 ) and refined with CNS ( Brünger et al. 1998 ). Since two monomers in the unit cell are related by a fractional translation along c of approximately 0.54, the intensities of the diffraction pattern are modulated by a factor of cos 2  (0.54πl). As a result, reflections with l-indices such that cos 2  (0.54πl) < ½ are systematically weak, leading to an R-factor higher than would be expected for a nonpseudocentered crystal structure. However, when only the reflections with cos 2  (0.54πl) > ½ (which will have a more normal intensity distribution) are used for the R-factor calculation, reasonable values for R are obtained. The geometry of the final model was analyzed with PROCHECK ( Morris et al. 1992 ). The Ramachandran plot shows 98.9% of the residues in the allowed regions and 1.1% in the disallowed regions. The main chain of K66, which constitutes the residue in the disallowed region, was modeled on segments taken from well-refined, high-resolution structures. The Protein Data Bank was searched for structural neighbors of AfJAMM using the DALI server ( Holm and Sander 1993 ). The superpositions with cytidine deaminase (1JTK), thermolysin (1FJQ), and ScNP (1C7K) were done using the LSQKAB program of the CCP4 distribution (CCP4 1994). All structural figures were made with PyMOL ( DeLano 2000 ).The experiments with  S. pombe  were performed as previously described by  Cope et al. (2002 ).
Materials and Methods
The gene for  A. fulgidus  JAMM ( Ponting et al. 1999 ), open reading frame  AF2198 , was cloned from genomic DNA (ATCC #49558D; American Type Culture Collection, Manassas, Virginia, United States) into the pCRT7 vectors (Invitrogen, Carlsbad, California, United States). During cloning, the alternate start codon, GTG, was replaced with the canonical start codon, ATG. The construct was expressed in BL21(DE3)pLysS cells (Novagen, Madison, Wisconsin, United States). The cells were grown to midlog phase in terrific broth media and induced with 0.5 mM IPTG. The cells were lysed by sonication and the protein was isolated by immobilized metal ion chromatography using a Ni-NTA resin (Qiagen, Valencia, California, United States). The protein was further purified by gel filtration on a Sephacryl S100 column (Amersham Pharmacia Biotech, Chalfont St Giles, United Kingdom) and concentrated. The amino-terminal tag was removed by limited digestion with trypsin. Mass spectrometry analysis revealed that trypsin only cut AfJAMM in the amino-terminal tag region, and only a single band was evident on a Coomassie-stained polyacrylamide gel. The tag and uncut protein were removed with Ni-NTA resin followed by anion-exchange chromatography with SOURCE 30Q resin (Amersham Pharmacia Biotech). The processed protein was then concentrated to approximately 30 mg/ml by ultrafiltration. The selenomethionine protein was produced as described elsewhere ( Van Duyne et al. 1993 ) and purified using the same protocol as for the native protein.AF2198Protein crystals were obtained in 100 mM NH 4 H 2 PO 4 , 200 mM sodium citrate (pH 5) using vapor diffusion with sitting drops and hanging drops. Crystals were incubated for approximately 1 min in a cryo-solution of equal volumes of reservoir solution and 35% meso-erythritol for the selenomethionine crystals and supplemented with 5 mM ZnCl 2  for the native crystals. The crystals belonged to the space group P6 5 , with cell dimensions of a = b = 76 Å, c = 94 Å and two subunits per asymmetric unit. Data for the selenomethionine crystals were collected on Beamline 9.2 at the Stanford Synchrotron Radiation Laboratory (SSRL) (Stanford, California, United States) and data for the native crystals were collected on Beamline 8.2.1 at the Advanced Light Source (ALS) (Lawrence Berkeley National Laboratory, Berkeley, California, United States) (see  Table 1 ).Phases were obtained by the MAD technique using data collected from selenomethionine-substituted crystals (see  Table 1 ). Three Se atoms were located by SOLVE ( Terwilliger and Berendzen 1999 ) and used to calculate the initial phases. Phasing was subsequently improved by noncrystallographic symmetry averaging, using operators derived from the Se positions, and solvent flattening in RESOLVE ( Terwilliger 2000 ). The polypeptide model was built in O ( Jones et al. 1991 ) and refined with CNS ( Brünger et al. 1998 ).Since two monomers in the unit cell are related by a fractional translation along c of approximately 0.54, the intensities of the diffraction pattern are modulated by a factor of cos 2  (0.54πl). As a result, reflections with l-indices such that cos 2  (0.54πl) < ½ are systematically weak, leading to an R-factor higher than would be expected for a nonpseudocentered crystal structure. However, when only the reflections with cos 2  (0.54πl) > ½ (which will have a more normal intensity distribution) are used for the R-factor calculation, reasonable values for R are obtained.The geometry of the final model was analyzed with PROCHECK ( Morris et al. 1992 ). The Ramachandran plot shows 98.9% of the residues in the allowed regions and 1.1% in the disallowed regions. The main chain of K66, which constitutes the residue in the disallowed region, was modeled on segments taken from well-refined, high-resolution structures. The Protein Data Bank was searched for structural neighbors of AfJAMM using the DALI server ( Holm and Sander 1993 ). The superpositions with cytidine deaminase (1JTK), thermolysin (1FJQ), and ScNP (1C7K) were done using the LSQKAB program of the CCP4 distribution (CCP4 1994). All structural figures were made with PyMOL ( DeLano 2000 ).The experiments with  S. pombe  were performed as previously described by  Cope et al. (2002 ).Supporting Information Accession Numbers The accession numbers for the proteins discussed in this paper are 20S proteasomes (PDB ID 1RYP), AfJAMM (Entrez Protein ID NP_071023; PDB ID 1R5X), AMSH (Entrez Protein ID NP_006454), AtCSN5/AJH1 (Entrez Protein ID NP_173705), AtRpn11 (Entrez Protein ID NP_197745), C6.1A (Entrez Protein ID NP_077308), CeCSN5 (Entrez Protein ID NP_500841), CeRpn11 (Entrez Protein ID NP_494712), Csn5 (Entrez Protein ID NP_593131), Cul1 (Entrez Protein ID NP_594259), cytidine deaminase (PDB ID 1JTK), DmCsn5/CH5 (Entrez Protein ID NP_477442), DmRpn11/p37b (Entrez Protein ID AAF08394), EcRadC (Entrez Protein ID NP_418095), HsAMSH (Entrez Protein ID NP_006454), HsC6.1A (Entrez Protein ID NP_077308), HsCsn5 (Entrez Protein ID NP_006828), HsRpn11/POH1 (Entrez Protein ID NP_005796), JAB1 (Entrez Protein ID AAC17179), lambdaK (Entrez Protein ID AAA96551), Mov34 (Entrez Protein ID NP_034947), Mpr1p (Entrez Protein ID AAN77865), Nedd8 (Swiss-Prot ID Q15843), neurolysin (PDB ID 1I1I), Pad1p (Entrez Protein ID NP_594014), phage λ tail assembly protein K (Entrez Protein ID AAA96551), RadC (Entrez Protein ID NP_418095), Rpn11 (Entrez Protein ID AAN77865), SCF (PDB ID 1LDK), ScNP (PDB ID 1C7K), ScRpn11 (Entrez Protein ID AAN77865), Sic1 (Entrez Protein ID 1360441), SpCsn5 (Entrez Protein ID NP_593131), SpRpn11/Pad1 (Entrez Protein ID NP_594014), thermolysin (PDB ID 1FJQ), ubiquitin (Swiss-Prot ID P04838), UBP (PDB ID 1NB8), and UCH (PDB ID 1UCH). These databases may be found at  http://www.ncbi.nlm.nih.gov/entrez/  (Entrez Protein),  http://www.rcsb.org/pdb/  (Protein Data Bank [PDB]), and  http://us.expasy.org/sprot/  (Swiss-Prot).
Supporting Information
Accession Numbers The accession numbers for the proteins discussed in this paper are 20S proteasomes (PDB ID 1RYP), AfJAMM (Entrez Protein ID NP_071023; PDB ID 1R5X), AMSH (Entrez Protein ID NP_006454), AtCSN5/AJH1 (Entrez Protein ID NP_173705), AtRpn11 (Entrez Protein ID NP_197745), C6.1A (Entrez Protein ID NP_077308), CeCSN5 (Entrez Protein ID NP_500841), CeRpn11 (Entrez Protein ID NP_494712), Csn5 (Entrez Protein ID NP_593131), Cul1 (Entrez Protein ID NP_594259), cytidine deaminase (PDB ID 1JTK), DmCsn5/CH5 (Entrez Protein ID NP_477442), DmRpn11/p37b (Entrez Protein ID AAF08394), EcRadC (Entrez Protein ID NP_418095), HsAMSH (Entrez Protein ID NP_006454), HsC6.1A (Entrez Protein ID NP_077308), HsCsn5 (Entrez Protein ID NP_006828), HsRpn11/POH1 (Entrez Protein ID NP_005796), JAB1 (Entrez Protein ID AAC17179), lambdaK (Entrez Protein ID AAA96551), Mov34 (Entrez Protein ID NP_034947), Mpr1p (Entrez Protein ID AAN77865), Nedd8 (Swiss-Prot ID Q15843), neurolysin (PDB ID 1I1I), Pad1p (Entrez Protein ID NP_594014), phage λ tail assembly protein K (Entrez Protein ID AAA96551), RadC (Entrez Protein ID NP_418095), Rpn11 (Entrez Protein ID AAN77865), SCF (PDB ID 1LDK), ScNP (PDB ID 1C7K), ScRpn11 (Entrez Protein ID AAN77865), Sic1 (Entrez Protein ID 1360441), SpCsn5 (Entrez Protein ID NP_593131), SpRpn11/Pad1 (Entrez Protein ID NP_594014), thermolysin (PDB ID 1FJQ), ubiquitin (Swiss-Prot ID P04838), UBP (PDB ID 1NB8), and UCH (PDB ID 1UCH). These databases may be found at  http://www.ncbi.nlm.nih.gov/entrez/  (Entrez Protein),  http://www.rcsb.org/pdb/  (Protein Data Bank [PDB]), and  http://us.expasy.org/sprot/  (Swiss-Prot).
Accession Numbers
The accession numbers for the proteins discussed in this paper are 20S proteasomes (PDB ID 1RYP), AfJAMM (Entrez Protein ID NP_071023; PDB ID 1R5X), AMSH (Entrez Protein ID NP_006454), AtCSN5/AJH1 (Entrez Protein ID NP_173705), AtRpn11 (Entrez Protein ID NP_197745), C6.1A (Entrez Protein ID NP_077308), CeCSN5 (Entrez Protein ID NP_500841), CeRpn11 (Entrez Protein ID NP_494712), Csn5 (Entrez Protein ID NP_593131), Cul1 (Entrez Protein ID NP_594259), cytidine deaminase (PDB ID 1JTK), DmCsn5/CH5 (Entrez Protein ID NP_477442), DmRpn11/p37b (Entrez Protein ID AAF08394), EcRadC (Entrez Protein ID NP_418095), HsAMSH (Entrez Protein ID NP_006454), HsC6.1A (Entrez Protein ID NP_077308), HsCsn5 (Entrez Protein ID NP_006828), HsRpn11/POH1 (Entrez Protein ID NP_005796), JAB1 (Entrez Protein ID AAC17179), lambdaK (Entrez Protein ID AAA96551), Mov34 (Entrez Protein ID NP_034947), Mpr1p (Entrez Protein ID AAN77865), Nedd8 (Swiss-Prot ID Q15843), neurolysin (PDB ID 1I1I), Pad1p (Entrez Protein ID NP_594014), phage λ tail assembly protein K (Entrez Protein ID AAA96551), RadC (Entrez Protein ID NP_418095), Rpn11 (Entrez Protein ID AAN77865), SCF (PDB ID 1LDK), ScNP (PDB ID 1C7K), ScRpn11 (Entrez Protein ID AAN77865), Sic1 (Entrez Protein ID 1360441), SpCsn5 (Entrez Protein ID NP_593131), SpRpn11/Pad1 (Entrez Protein ID NP_594014), thermolysin (PDB ID 1FJQ), ubiquitin (Swiss-Prot ID P04838), UBP (PDB ID 1NB8), and UCH (PDB ID 1UCH).These databases may be found at  http://www.ncbi.nlm.nih.gov/entrez/  (Entrez Protein),  http://www.rcsb.org/pdb/  (Protein Data Bank [PDB]), and  http://us.expasy.org/sprot/  (Swiss-Prot).