plbi-02-01-02 113..119

JAMM: A Metalloprotease-Like Zinc Site

in the Proteasome and Signalosome

Xavier I. Ambroggio

, Douglas C. Rees

2,3

, Raymond J. Deshaies

1,3

Division of Biology, California Institute of Technology, Pasadena, California, United States of America,

Division of Chemistry and Chemical Engineering, California Institute

of Technology, Pasadena, California, United States of America,

Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America

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

HS/

THX

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

subunits—which contain the proteo-

lytic active sites responsible for the protein degradation

activity of the proteasome—flanked by heptameric rings of

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 ubiquiti-

nated 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

HS/THX

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

þ

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 sub-

strate 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 metal-

loprotease architecture.

Received August 29, 2003; Accepted October 9, 2003; Published November 24,

2003

DOI: 10.1371/journal.pbio.0020002

Ó

2003 Ambroggio et al. This is an open-access article distributed

under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the

original work is properly cited.

Abbreviations: AfJAMM,

A. fulgidus

JAMM protein; AMSH, associated molecule with

SH3 domain of STAM; CDA, cytidine deaminase; CSN, COP9 signalosome; Cul1,

Cullin 1; DUB, deubiquitinating enzyme; JAMM, JAB1/MPN/Mov34 metalloenzyme;

MAD, multiwavelength anomalous diffraction; MPN, Mpr1p Pad1p N-terminal

domain; Nedd8, neural precursor cell expressed, developmentally downregulated

8; RMS, root-mean squared; Rpn11, regulatory particle number 11; SCF, Skp1/

Cdc53/Cullin/F-box receptor; ScNP,

S. caespitosus

zinc endoprotease; Ub, ubiquitin;

Ubls, ubiquitin-like proteins; UBP, ubiquitin-specific protease; UCH, ubiquitin C-

terminal hydrolase

Academic Editor: Hidde L. Ploegh, Harvard Medical School

*To whom correspondence should be addressed. E-mail: deshaies@caltech.edu

PLoS Biology | http://biology.plosjournals.org

January 2004 | Volume 2 | Issue 1 | Page

0113

BIOLOGY

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.

DOI: 10.1371/journal.pbio.0020002.g001

Table 1.

Data Collection Statistics

Native (Zn

þ

)

Selenomethionine MAD

Crystal 1

Crystal 2

Peak

Inflection

Remote

Peak (2)

Beamline

ALS 8.2.1

SSRL 9.2

Wavelength (A

)

1.1271

0.9790

0.9792

0.9184

0.9790

Resolution (A

)

38-2.3

30-2.5

30-2.3

sym

(%)

9.4 (37.9)

7.5 (43.9)

6.8 (35.7)

6.9 (40.0)

7.3 (42.8)

Completeness (%)

99.9 (99.9)

100 (100)

99.9 (99.8)

100 (100)

4.7 (1.8)

20.0 (3.4)

13.9 (2.5)

14.0 (2.6)

34.0 (6.1)

All reflections

278,220

111,979

55,743

56,206

285,937

Redundancy

20.6

10.5

5.2

5.3

21.1

Refinement

cryst

free

(%)

26.1/30.4

N-cryst

N-free

(%)

22.6/27.0

Number of protein atoms

1634

Number of Zn

þ

atoms

Number of waters

RMS deviation bonds (A

)

0.006

RMS deviation angles (

)

1.21

Owing to pseudocentering, reflections with l values such that cos

(0.54

½ are systematically weak, leading to an R-factor higher than would be expected for a

nonpseudocentered crystal structure. R

are the R-factors calculated with only the reflections with cos

(0.54

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.

DOI: 10.1371/journal.pbio.0020002.t001

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January 2004 | Volume 2 | Issue 1 | Page

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JAMM: A Metalloprotease-Like Zinc Site

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

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).

AfJAMM consists of an eight-stranded

sheet (

1–

8),

flanked by a long

helix (

1) between the first and second

strand, and a short

helix (

2) between the fourth and fifth

strand. This

sheet resembles a

barrel halved longitudinally

and curled around

1 (Figure 2A). The

2 helix is oriented

lengthwise on the convex surface of the

sheet. The zinc-

binding site is adjacent to a loop that spans the end of

4to

the beginning of

2 and is stabilized by a disulfide bond

between C74 from this loop to C95 on

5. 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 A

over 79

carbons, despite only 9% sequence identity

over structurally aligned residues. The catalytic zinc ions of

AfJAMM and CDA, 4.9 A

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 2A). Consequently, the JAMM fold repre-

sents 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) (John-

ston 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

sheet formed at the dimer

interface (Figure 2B). The subunits are related by a 2-fold

screw axis along the crystallographic c-axis with a translation

of 3.38 A

, corresponding to a displacement of one residue

along the

3 strand. AfJAMM behaves as a monomer during

size exclusion chromatography, suggesting that the dimer

observed in the asymmetric unit is an artifact of crystalliza-

tion. Yet the residues of

3 are highly conserved among

JAMM proteins (see Figure 1) and predominantly hydro-

phobic, which makes it difficult to regard the observed

interaction as completely insignificant. Flanking

3 to the

carboxy-terminal side, there is a striking covariation of

residues, MPQSGTG in Rpn11 orthologues and LPVEGTE

in Csn5 orthologues. The potential of

3 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

2–

4 sheet and

2. The

catalytic zinc has a tetrahedral coordination sphere (Figure

3A), with ligands provided by N

of H67 and H69 on

4, the

carboxylate of D80 on

2, and a water molecule. The latter

hydrogen-bonds to the sidechain of E22 on

2. 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 A

fJAMM and eukaryotic JAMM

homologues is still unknown. The majority of metallopro-

teases naturally employ zinc but show altered activities with

other substituted metals (Auld 1995).

The arrangement of zinc ligands in AfJAMM resembles that

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

1–

and

1–

8. 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 A

DOI: 10.1371/journal.pbio.0020002.g002

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January 2004 | Volume 2 | Issue 1 | Page

0115

JAMM: A Metalloprotease-Like Zinc Site

found in thermolysin, the

Streptomyces caespitosus

zinc endo-

protease (ScNP), and neurolysin, a mammalian metallopro-

tease (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 A

(Figure 3B).

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

hydroxyl group of the highly conserved S77 in AfJAMM

occupies a position similar to N

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 muta-

genesis of

Schizosaccharomyces pombe csn5

þ

. 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

þ

background (Figure 4A). The E56A mutation had no effect on

the assembly of Csn5 with Csn1

myc13

, while assembly with

S128A was slightly hindered (Figure 4A). 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 phe-

notype in

csn5

þ

cells (Figure 4B).

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

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

) colored in red. The aqua ligand, which lies at 2.9 A

from the zinc, is shown as a

red sphere surrounded by purple density (contoured at 3

)ofanF

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 A

from the zinc. The N

atoms

of H67 and H69 lie 2.1 A

from the zinc. The carboxylate oxygen atoms of E22 lie 3.2–3.5 A

from the aqua ligand and 4.5–5.0 A

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

of S77 in AfJAMM, O

of Y95 in ScNP, and the N

of H231 in thermolysin.

DOI: 10.1371/journal.pbio.0020002.g003

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JAMM: A Metalloprotease-Like Zinc Site

proteins (Figure 4C) such as AMSH and C6.1A, as well as the

prokaryotic protein RadC and the viral phage

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 impli-

cations of the AfJAMM structure explain why the deubiquiti-

nating 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 hydrox-

amate 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 protea-

some 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 polyacryla-

mide 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

, 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

for the native crystals. The crystals belonged to the space

group P6

, with cell dimensions of a

¼

¼

76 A

¼

94 A

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

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

csn5

D

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).

canonical JAMM motif residues are highlighted in green. The

conserved proline is highlighted in blue, and semiconserved cysteine

is highlighted in yellow.

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JAMM: A Metalloprotease-Like Zinc Site

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 oper-

ators 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 (Bru

nger 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

(0.54

l). As a

result, reflections with l-indices such that cos

(0.54

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

(0.54

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 deam-

inase (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

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).

Acknowledgments

This work was supported by the National Science Foundation

Graduate Research Fellowship and the Gordon Moore Foundation

(to XIA), as well as the Howard Hughes Medical Institute (to DCR and

RJD). We would like to thank the staff at the Stanford Synchrotron

Radiation Laboratory, a national user facility operated by Stanford

University on behalf of the United States Department of Energy,

Office of Basic Energy Sciences, and the Advanced Light Source,

which is supported by the Director of the Office of Science, Office of

Basic Energy Sciences, Materials Sciences Division of the United

States Department of Energy under contract number DE-AC03-

76SF00098 at Lawrence Berkeley National Laboratory. Special thanks

go to R. Verma and G. Cope for assistance with the associated

biochemistry, T. Yeates and O. Einsle for insights concerning the

treatment of the pseudocentered crystals, K. Locher and P. Strop for

helpful discussions, and J. Ambroggio for back massages and constant

support.

Conflicts of interest.

The authors have declared that no conflicts of

interest exist.

Author contributions.

XIA, DCR, and RJD conceived and designed

the experiments. XIA performed the experiments. XIA, DCR, and

RJD analyzed the data. XIA, DCR, and RJD contributed reagents/

materials/analysis tools. XIA wrote the paper.

&

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