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Protein Science (2006), 15:1182-1186. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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PROTEIN STRUCTURE REPORT

Structure of the Sir3 protein bromo adjacent homology (BAH) domain from S. cerevisiae at 1.95 Å resolution

Zhonggang Hou, John R. Danzer, Catherine A. Fox1 and James L. Keck1

Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706-1532, USA

(RECEIVED December 22, 2005; FINAL REVISION February 2, 2006; ACCEPTED February 13, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
Sir3p is a silent-information-regulator (SIR) protein required for the assembly of a transcriptionally "silent" chromatin structure at telomeres and the cryptic HM mating-type loci in Saccharomyces cerevisiae. Sir3p contains a putative "bromo adjacent homology" (BAH) domain at its N terminus that shares strong sequence similarity with the BAH domain of a subunit of the origin recognition complex (ORC), Orc1p. The Orc1p–BAH domain forms a well-defined complex with the ORC interaction region (OIR) of another Sir protein, Sir1p, which targets formation of silent chromatin to the HM-loci. Interestingly, despite sequence similarity of the Sir3p and Orc1p BAH domains and Sir3p's established importance in silencing, Sir3p does not bind the Sir1p–OIR. Here we report the 1.95 Å resolution crystal structure of the Sir3p–BAH domain. The structure reveals two key features that can account for Sir3p–BAH domain's inability to interact with Sir1p. First, several Orc1p–BAH domain residues known to directly contact Sir1p are altered in the Sir3p–BAH domain. Second, a critical OIR-binding pocket present on the surface of the Orc1p–BAH domain is "filled" in the Sir3p–BAH domain structure, potentially making it inaccessible to Sir1p. These findings imply that the Sir3p–BAH domain structure has evolved for functions distinct from those of the Orc1p–BAH domain.

Keywords: Sir; ORC; silencing


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Transcriptional silencing at the cryptic mating-type loci, HMR and HML, and telomeres in the yeast Saccharomyces cerevisiae has served as a paradigm for the formation of specialized domains of chromatin that regulate gene expression. Silencing is a form of transcriptional repression that requires the formation of a long-range repressive (silent) chromatin structure analogous to heterochromatin in metazoans (Rusche et al. 2003). Sir3p is one of three Sir (silent information regulator) proteins that plays a critical role in the assembly and structure of silent chromatin. Recent evidence has shown that Sir3p functions in a dynamic complex with two other Sir proteins, Sir2 and Sir4 (Liou et al. 2005), and helps this complex modify histones through the NAD-dependent protein deacetylase activity of Sir2p (Denu 2003; Moazed et al. 2004). The Sir2–4 complex then binds directly to the deacetylated nucleosomes, in part through favorable interactions between Sir3 and deacetylated histones (Carmen et al. 2002), thereby becoming a structural component of a higher order silent chromatin domain at the HM and telomeric loci (Rusche et al. 2003).

Although it is clear that Sir3p must interact with other Sir proteins to function in silencing, molecular and structural details about these interactions are lacking, and the mechanisms underlying Sir3’s functions in silencing are unknown. At the level of primary structure, Sir3p is most similar to Orc1p, which has well-established roles in both DNA replication and transcriptional silencing. Orc1p is the largest subunit of the origin recognition complex (ORC), a conserved six-subunit heteromeric protein complex that binds replication origins and is essential for the initiation of DNA replication in all eukaryotes (Bell 2002). The primary role for ORC in silencing the HM loci is to bind Sir1p, a HM-specific Sir protein that, in turn, helps recruit and/or stabilize the Sir2–4 complex(es) at these loci (Fox and McConnell 2005). Physical association between Orc1p and Sir1p occurs between discreet domains within each protein: the ORC interaction region (OIR) of Sir1p (Bose et al. 2004) and the N-terminal bromo adjacent homology (BAH) domain of Orc1p (Zhang et al. 2002). The Orc1p BAH domain is required for Orc1p's role in silencing but is dispensable for its essential function in DNA replication (Bell et al. 1995). High-resolution crystal structures of the OIR and the Orc1p–BAH domains and of the complex formed between these domains have been solved recently, providing structural insights into a protein–protein interaction that is critical to the establishment of silent chromatin (Zhang et al. 2002; Hou et al. 2005; Hsu et al. 2005).

Sir3’s significant similarity to Orc1p and its shared biological role with both Orc1p and Sir1p in silencing raise the reasonable postulate that it may also form a complex with Sir1p important for silencing. Sir3p and Orc1p are strikingly similar over their N-terminal ~216 amino acids, sharing 48% identity and 67% similarity (Bell et al. 1995; Fig. 1A). However, directed two-hybrid assays under conditions that revealed the robust interaction between Sir1p and the Orc1p–BAH domain failed to detect a Sir3p/Sir1p interaction (Triolo and Sternglanz 1996), and GST pull-down experiments also failed to detect a Sir3p–BAH/Sir1p interaction under conditions that allowed for a robust Orc1p–BAH/Sir1p interaction (Zhang et al. 2002). In addition, genetic evidence argues against a simple Sir1p-binding role for the Sir3p–BAH domain in silencing. For example, Sir1p is dispensable for telomeric silencing (Fox et al. 1997), yet mutational analysis of SIR3 supports a role for the Sir3p–BAH domain in telomeric silencing and for efficient HML-silencing in cells harboring a sir1{Delta} mutation (Stone et al. 2000). Together, these data support the conclusion that the Sir3p–BAH domain's role in silencing does not involve interactions with the Sir1p–OIR despite the intriguing and significant similarity between the Orc1p and Sir3p BAH domains.


Figure 1
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  		Figure 1. (A) Structure-based sequence alignment between the Sir3p and Orc1p BAH domains. beta-Strands and {alpha}-helices are diagrammed as arrows and boxes, respectively, with regions lacking electron-density shown as dots. The lines under the Orc1p–BAH sequence indicate amino acids involved in direct contact with the Sir1p–OIR in the complex formed between these domains (Hou et al. 2005; Hsu et al. 2005). The asterisk above amino acids in the Sir3p–BAH sequence denotes residues implicated in the in vivo functions of Sir3p based on genetic analyses (Johnson et al. 1990; Liu and Lustig 1996; Stone et al. 2000; Wang et al. 2004). (B) Stereo diagram of the Sir3p–BAH domain. Secondary structural elements from A, and the N and C termini are labeled. (C) Least-squares superimposition of the Orc1p (green) and Sir3p BAH (red) domain structures. The H domain is indicated.

 
Here, we define the 1.95 Å resolution structure of the Sir3p–BAH domain. Our structure reveals this domain's striking secondary and tertiary structural similarity with the Orc1p–BAH domain as predicted based on their sequence similarity. However, comparison of surface features between the Sir3p–BAH and Orc1p–BAH domains shows that the Sir3p–BAH domain lacks several key features that stabilize the complex that forms between the Sir1p–OIR and Orc1p–BAH domains.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
In the course of purifying the S. cerevisiae Sir3p–BAH domain (residues 1–229 of Sir3p) for biochemical studies, we discovered that it formed small crystals spontaneously during dialysis. Crystallization trials of the Sir3p–BAH led to the formation of crystals that diffracted to 1.95 Å resolution (Table 1). We determined the structure of Sir3p–BAH by molecular replacement using the Orc1p–BAH domain structure (Zhang et al. 2002) as a search model followed by rounds of manual fitting and refinement. The crystal form contained one molecule per asymmetric unit, and the final model included residues 8–215, excluding residues 24–36, 77–81, and 159–165, for which electron density was not observed. The excluded residues lay in exposed loops in the structure.


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  		Table 1. Data collection and refinement statistics

 
The Sir3p–BAH domain forms a globular oval-shaped structure with dimensions of 30 x 30 x 60 Å and exhibits the characteristic core of intertwined beta-sheets that defines the BAH structural domain (Fig. 1B). Least-squares superimposition of the Sir3p–BAH domain structure with that from the Orc1p reveals their highly conserved structural features (root mean square deviation [RMSD] = 1.6 Å for 180 common C{alpha} atoms; see Fig. 1C). This structural similarity is not surprising given the high level of amino acid similarity between these two protein domains (Fig. 1A). Notably, both domains contain similar extruded "H-domains," which, in the Orc1p–BAH domain, forms a key portion of the essential interface with Sir1p (Hou et al. 2005; Hsu et al. 2005). The H domain is comprised of three helices ({alpha}2, {alpha}3, and {alpha}4) that are not found in other BAH domain proteins (Zhang et al. 2002). Significantly, the differences in their H-domains are sufficient for explaining the biochemical differences between the Orc1p–BAH and Sir3p–BAH domains in terms of binding Sir1p; a recombinant chimeric protein, in which the H-domain of Sir3p was replaced with that of Orc1p, is able to bind the C-terminal amino acids 348–678 of Sir1p in a GST pull-down experiment (Zhang et al. 2002).

A more detailed examination of the surface structure of the Sir3p–BAH domain, particularly regions formed by the H domain, revealed a number of molecular features that could account for these observations. First, although the H domains of the two proteins overlapped substantially (RMSD = 1.6 Å for 31 C{alpha} atoms), several amino acid differences existed between the two domains in regions known to form elements of the Orc1p/Sir1p interface (Fig. 1A, regions underlined in Orc1p). For example, in the {alpha}4 helix of Orc1p–BAH, four amino acids are important for its interaction with the Sir1p–OIR: N120, K121, F123, and S124. Of these four residues, only F123 is invariant between Sir3p–BAH and Orc1p–BAH, with substitutions of K120, D121, and F124 found in the Sir3p–BAH domain (Fig. 1A). In addition, five residues— E61, A62, A63, G64, and T65—that comprise the loop between the beta4 and beta5 strands and the N terminus of beta5 form another surface on Orc1p that interacts with the Sir1p–OIR (Fig. 1). Of these five residues, only one, T65, is conserved in the Sir3p–BAH domain. Thus, although the secondary structural features of the Sir3p–BAH and Orc1p–BAH domains showed remarkable similarity, there are differences in key surface residues important for forming a complex with Sir1p–OIR.

A second difference that is likely to help explain biochemical differences between the BAH domains became apparent when we examined their surface representations (Fig. 2). The analogous surfaces on both protein domains formed a superficially similar overall concave surface, although the width and the depth of these regions differed somewhat (Fig. 2A,B). The high-resolution structure of the complex between the Orc1p–BAH and Sir1p–OIR domains indicates that such differences are likely significant; the Orc1p–BAH surface that binds Sir1p–OIR is highly complementary to the convex interaction surface presented by the Sir1p–OIR (Hou et al. 2005; Hsu et al. 2005). A particularly striking example of this significance is provided by a major contact point between the Orc1p–BAH and Sir1p–OIR that is formed by the insertion of P492 from Sir1p into a deep, hydrophobic pocket on the Orc1p–BAH domain formed by a cluster of six amino acids on the Orc1p–BAH surface (Fig. 2B). This pocket is notably absent in Sir3p–BAH (Fig. 2A). Interestingly, and in contrast to the examples discussed above, several of the amino acids in Orc1p that form this pocket are either invariant or chemically similar to the analogous amino acids in Sir3p (Fig. 2C). Specifically, W93, F94, F123, and P98 are invariant between Orc1p and Sir3p, and V96 and N120 of Orc1p are conservatively substituted with L96 and K120 in Sir3p. However, in this Sir1p–OIR contact point, the single nonconservative substitution of S124 on Orc1p with F124 on Sir3p is sufficient for burying a potential binding pocket, creating instead a relatively flat surface that would prevent Sir1p P492 from inserting into the pocket (Fig. 2A). Such an effect would also preclude the rest of the Sir1p–OIR interaction surface from fitting snugly into the analogous interaction surface on the Sir3 BAH domain.


Figure 2
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  		Figure 2. (A) Orthogonal views of a surface representation of the Sir3p–BAH domain in which amino acids that align with the Sir1p–OIR-interacting surface in the Orc1p–BAH domain are colored blue. (B) Orthogonal views of a surface representation of the Orc1p–BAH domain in which amino acids that form the Sir1p–OIR-interacting surface are colored blue. The P492 of Sir1p that binds in a deep pocket formed on this surface is shown as a semitransparent orange surface. (C) Close-up view of the residues within the Sir1p–P492 binding pocket in the Orc1p–BAH domain (ball-and-stick form, right) compared to the analogous region in Sir3p–BAH (left). The phenylalanine residue colored red in the Sir3p–BAH domain is buried in the hydrophobic core of the domain; in contrast, the analogous residue in the Orc1p–BAH is partially solvent-exposed.

 
In summary, the structure of the Sir3p–BAH domain presented here provides compelling structural explanations for why this domain, despite its sequence similarity to the Orc1p–BAH domain-binding partner of Sir1p, failed to interact substantially with the Sir1p–OIR. However, these data also clearly indicated that the Sir3p–H-domain, like the Orc1p–H-domain, contributes to a large potential interaction surface, making it tempting to speculate that the Sir3p–BAH domain might also have an as yet undiscovered partner important for transcriptional silencing. Genetic and molecular analyses hint that this partner, in contrast to the Sir1p partner of the Orc1p–BAH domain, would have functions in both telomeric and HM silencing and that, unlike the Orc1p–BAH domain, the Sir3p–BAH domain would not have a separable and specific role in HM silencing (Johnson et al. 1990; Bell et al. 1995; Liu and Lustig 1996; Wade et al. 1997; Stone et al. 2000). Further genetic analyses are needed to test this hypothesis.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
Expression and purification of the Sir3p–BAH domain
The region encoding Sir3p amino acids 1–229 was amplified by PCR and introduced into the NheI site of pET28b (Novagen) creating pET28b–(HIS)6–SIR3–BAH. This plasmid was introduced into the Escherichia coli strain Rosetta (DE3) pLysS (Novagen), and the cells were grown in Luria–Bertani medium supplemented with 30 µg/mL chloramphenicol and 50 µg/mL kanamycin at 37°C. At OD600nm = 0.5, cells were shifted to 30°C and expression of (His)6–Sir3p–BAH induced with 1 mM isopropyl beta-D-thiogalactopyranoside for 3 h. Cells were harvested by centrifugation, frozen at –80°C, resuspended at 4°C in lysis buffer (50 mM HEPES at pH 6.5, 500 mM sodium chloride, 10% glycerol, 20 mM imidazole) supplemented with 10 µg/mL DNaseI (Sigma), and lysed by sonication on ice. The resulting extract was cleared by centrifugation (38,000g for 15 min) and incubated with Ni-NTA resin (Novagen) at 4°C for 1 h. The resin was then packed into a column and washed with 10 column volumes of lysis buffer, and (His)6–Sir3p–BAH was eluted with elution buffer (lysis buffer plus 500 mM Imidazole). The (His)6–Sir3p–BAH was buffer-exchanged into lysis buffer with 60 units of thrombin (Sigma) to remove the (His)6 N-terminal tag. The cleaved Sir3p–BAHp was then flowed over a second Ni-NTA column, concentrated, and purified over a Sephacryl S-100 column (Amersham Biosciences) equilibrated with 50 mM HEPES (pH 6.5), 500 mM sodium chloride, 10% glycerol. Protein purity was determined to be >95% by SDS-PAGE. Protein concentrations were measured spectrophotometrically by measuring sample A280nm in 6.0 M Guanidine-HCl and using a calculated molar extinction coefficient of 40,090 mol–1 cm–1.

Crystallization and structure determination of Sir3p–BAH
Purified Sir3p-BAH was concentrated to 6–8 mg/mL and dialyzed into 10 mM HEPES (pH 6.5), 50 mM sodium chloride. Crystals appeared spontaneously upon dialysis. To control crystal growth, Sir3p–BAH was dialyzed into 400 mM ammonium acetate prior to crystallization trials. Sir3p–BAH crystals formed under a variety of conditions, but final diffraction quality crystals were grown at room temperature by hanging-drop vapor diffusion mixing 1 µL of protein (6 mg/mL) with 1 µL of well solution (0.1 M HEPES at pH 6.5, 200 mM sodium chloride, 5%–15% PEG 400). Crystals were transferred to cryoprotection buffer (0.1 M HEPES at pH 6.5, 200 mM sodium chloride, 30% PEG 400) before freezing in liquid nitrogen for data collection. Crystals diffracted to 1.95 Å resolution with C2 symmetry and unit cell dimensions of a = 94.6 Å, b = 44.1 Å, c = 53.7 Å, {alpha} = {gamma} = 90, beta = 96.9°, consistent with one molecule of Sir3p–BAH per asymmetric unit (Matthews 1968). The final structure was determined by molecular replacement using AMORE (Navaza 2001) with the Orc1p–BAH structure (Zhang et al. 2002) as a search model. The molecular replacement solution was refined with REFMAC5_ARP (Winn et al. 2001) and by manually rebuilding the model with O (Jones et al. 1991).


   Footnotes
 
1 These authors contributed equally to this work. Back

Reprint requests to: James L. Keck or Catherine A. Fox, Department of Biomolecular Chemistry, 587 Medical Sciences Center, 1300 University Avenue, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706-1532, USA; e-mail: jlkeck{at}wisc.edu or cfox{at}wisc.edu; fax: (608) 262-5253.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.052061006.


   Acknowledgments
 
This work was supported by NIH Grant GM056890 (to C.A.F.), GM072392 (NRSA fellowship to J.R.D.), and Shaw Scientist Awards from the Milwaukee Foundation (to C.A.F. and J.L.K.). The atomic coordinates and structure factors for the Sir3 BAH domain (PDB ID 2FL7) have been deposited in the Protein Data Bank (http://www.rcsb.org).


   References
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 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 References
 
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Carmen A.A., Milne L., Grunstein M. 2002. Acetylation of the yeast histone H4 N terminus regulates its binding to heterochromatin protein SIR3 J. Biol. Chem. 277: 4778–4781.[Abstract/Free Full Text]

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