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Protein O -glycosylation is an important post-translational modification in all organisms, but deciphering the specific functions of these glycans is difficult due to their structural complexity. Understanding the glycosylation of mucin-like proteins presents a particular challenge as they are modified numerous times with both the enzymes involved and the glycosylation patterns being poorly understood. Here we systematically explored the O -glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogen Streptococcus pneumoniae TIGR4. Previous works have assigned the function of 3 of the 10 glycosyltransferases thought to modify PsrP, GtfA/B, and Gtf3 as catalyzing the first two reactions to form a unified disaccharide core structure. We now use in vivo and in vitro glycosylation assays combined with hydrolytic activity assays to identify the glycosyltransferases capable of decorating this core structure in the third and fourth steps of glycosylation. Specifically, the full-length GlyE and GlyG proteins and the GlyD DUF1792 domain participate in both steps, whereas full-length GlyA and the GlyD GT8 domain catalyze only the fourth step. Incorporation of different sugars to the disaccharide core structure at multiple sites along the serine-rich repeats results in a highly polymorphic product. Furthermore, crystal structures of apo- and UDP-complexed GlyE combined with structural analyses reveal a novel Rossmann-fold "add-on" domain that we speculate to function as a universal module shared by GlyD, GlyE, and GlyA to forward the peptide acceptor from one enzyme to another. These findings define the complete glycosylation pathway of a bacterial glycoprotein and offer a testable hypothesis of how glycosyltransferase coordination facilitates glycan assembly., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Invertases catalyze the hydrolysis of sucrose to glucose and fructose, thereby playing a key role in primary metabolism and plant development. According to the optimum pH, invertases are classified into acid invertases (Ac-Invs) and alkaline/neutral invertases (A/N-Invs), which share no sequence homology. Compared with Ac-Invs that have been extensively studied, the structure and catalytic mechanism of A/N-Invs remain unknown. Here we report the crystal structures of Anabaena alkaline invertase InvA, which was proposed to be the ancestor of modern plant A/N-Invs. These structures are the first in the GH100 family. InvA exists as a hexamer in both crystal and solution. Each subunit consists of an (α/α) 6 barrel core structure in addition to an insertion of three helices. A couple of structures in complex with the substrate or products enabled us to assign the subsites -1 and +1 specifically binding glucose and fructose, respectively. Structural comparison combined with enzymatic assays indicated that Asp-188 and Glu-414 are putative catalytic residues. Further analysis of the substrate binding pocket demonstrated that InvA possesses a stringent substrate specificity toward the α1,2-glycosidic bond of sucrose. Together, we suggest that InvA and homologs represent a novel family of glucosidases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375-Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a "T-shaped" pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a β-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides.

Saccharomyces cerevisiae Abz2 is a pyridoxal 5'-phosphate (PLP)-dependent lyase that converts 4-amino-4-deoxychorismate (ADC) to para-aminobenzoate and pyruvate. To investigate the catalytic mechanism, we determined the 1.9 Å resolution crystal structure of Abz2 complexed with PLP, representing the first eukaryotic ADC lyase structure. Unlike Escherichia coli ADC lyase, whose dimerization is critical to the formation of the active site, the overall structure of Abz2 displays as a monomer of two domains. At the interdomain cleft, a molecule of cofactor PLP forms a Schiff base with residue Lys-251. Computational simulations defined a basic clamp to orientate the substrate ADC in a proper pose, which was validated by site-directed mutageneses combined with enzymatic activity assays. Altogether, we propose a putative catalytic mechanism of a unique class of monomeric ADC lyases led by yeast Abz2.

The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6'P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6'P (a non-metabolizable analog of cellobiose-6'P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)8 TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr(126), Tyr(303), and Trp(338), at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser(424), Lys(430), and Tyr(432) of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite -1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family.

The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX(9)C or twin CX(3)C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.

The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the β(1,3)-linked galactose and N-acetylglucosamine (Galβ(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Å and its complex with galactose at 1.95 Å. At pH 5.5-8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)(8) TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C(P)) to Cys-62 and the resolving cysteine (C(R)) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Å resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C(P) and C(R). An intermolecular C(P)-C(R) disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.

The β-N-acetylhexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (GH20) catalyzes the hydrolysis of the β-N-acetylglucosamine (NAG) group from the nonreducing end of various glycoconjugates. The putative surface-exposed N-acetylhexosaminidase StrH/Spr0057 from Streptococcus pneumoniae R6 was proved to contribute to the virulence by removal of β(1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase and β-galactosidase, respectively. StrH is the only reported GH20 enzyme that contains a tandem repeat of two 53% sequence-identical catalytic domains (designated as GH20-1 and GH20-2, respectively). Here, we present the 2.1 Å crystal structure of the N-terminal domain of StrH (residues Glu-175 to Lys-642) complexed with NAG. It adopts an overall structure similar to other GH20 enzymes: a (β/α)(8) TIM barrel with the active site residing at the center of the β-barrel convex side. The kinetic investigation using 4-nitrophenyl N-acetyl-β-d-glucosaminide as the substrate demonstrated that GH20-1 had an enzymatic activity (k(cat)/K(m)) of one-fourth compared with GH20-2. The lower activity of GH20-1 could be attributed to the substitution of active site Cys-469 of GH20-1 to the counterpart Tyr-903 of GH20-2. A complex model of NAGβ(1,2)Man at the active site of GH20-1 combined with activity assays of the corresponding site-directed mutants characterized two key residues Trp-443 and Tyr-482 at subsite +1 of GH20-1 (Trp-876 and Tyr-914 of GH20-2) that might determine the β(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity toward the β(1,2)-linked β-N-acetylglucosides.

Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe(3+) and Mn(2+) coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (Ap(n)A) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/Ap(n)A hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/Ap(n)A and phosphodiester hydrolase.

The methionine S-sulfoxide reductase MsrA catalyzes the reduction of methionine sulfoxide, a ubiquitous reaction depending on the thioredoxin system. To investigate interactions between MsrA and thioredoxin (Trx), we determined the crystal structures of yeast MsrA/Mxr1 in their reduced, oxidized, and Trx2-complexed forms, at 2.03, 1.90, and 2.70 Å, respectively. Comparative structure analysis revealed significant conformational changes of the three loops, which form a plastic "cushion" to harbor the electron donor Trx2. The flexible C-terminal loop enabled Mxr1 to access the methionine sulfoxide on various protein substrates. Moreover, the plasticity of the Trx binding site on Mxr1 provides structural insights into the recognition of diverse substrates by a universal catalytic motif of Trx.

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C-C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 A resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical alpha/beta-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C-C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr(121), Tyr(229), and Asn(132), which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 A resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 A apart. The overall fold of API-A belongs to the beta-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu(87) and Lys(145), and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met(83) to P5' Ala(92), adopts a novel conformation with the Leu(87) completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys(141) to P5' Glu(150), binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.

By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.

Epstein-Barr virus is a human herpesvirus that causes infectious mononucleosis and lymphoproliferative malignancies. LMP1 (latent membrane protein-1), which is encoded by this virus and which is essential for transformation of B lymphocytes, acts as a constitutively active mimic of the tumor necrosis factor receptor (TNFR) CD40. LMP1 is an integral membrane protein containing six transmembrane segments and a cytoplasmic domain at the C terminus that binds to intracellular TNFR-associated factors (TRAFs). TRAFs are intracellular co-inducers of downstream signaling from CD40 and other TNFRs, and TRAF3 is required for activation of B lymphocytes by LMP1. Cytoplasmic C-terminal activation region 1 of LMP1 bears a motif (PQQAT) that conforms to the TRAF recognition motif PVQET in CD40. In this study, we report the crystal structure of this portion of LMP1 C-terminal activation region-1 (204PQQATDD210) bound in complex with TRAF3. The PQQAT motif is bound in the same binding crevice on TRAF3 where CD40 is bound, providing a molecular mechanism for LMP1 to act as a CD40 decoy for TRAF3. The LMP1 motif is presented in the TRAF3 crevice as a close structural mimic of the PVQET motif in CD40, and the intermolecular contacts are similar. However, the viral protein makes a unique contact: a hydrogen bond network formed between Asp210 in LMP1 and Tyr395 and Arg393 in TRAF3. This intermolecular contact is not made in the CD40-TRAF3 complex. The additional hydrogen bonds may stabilize the complex and strengthen the binding to permit LMP1 to compete with CD40 for binding to the TRAF3 crevice, influencing downstream signaling to B lymphocytes and contributing to dysregulated signaling by LMP1.

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 A resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ' interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.

Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-A resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.

Lymphotoxin-beta receptor (LTbetaR) and CD40 are members of the tumor necrosis factor family of signaling receptors that regulate cell survival or death through activation of NF-kappaB. These receptors transmit signals through downstream adaptor proteins called tumor necrosis factor receptor-associated factors (TRAFs). In this study, the crystal structure of a region of the cytoplasmic domain of LTbetaR bound to TRAF3 has revealed an unexpected new recognition motif, 388IPEEGD393, for TRAF3 binding. Although this motif is distinct in sequence and structure from the PVQET motif in CD40 and PIQCT in the regulator TRAF-associated NF-kappaB activator (TANK), recognition is mediated in the same binding crevice on the surface of TRAF3. The results reveal structurally adaptive "hot spots" in the TRAF3-binding crevice that promote molecular interactions driving specific signaling after contact with LTbetaR, CD40, or the downstream regulator TANK.

Phox homology (PX) domains have been recently identified in a number of different proteins and are involved in various cellular functions such as vacuolar targeting and membrane protein trafficking. It was shown that these modules of about 130 amino acids specifically binding to phosphoinositides and that this interaction is crucial for their cellular function. The yeast genome contains 17 PX domain proteins. One of these, Grd19p, is involved in the localization of the late Golgi membrane proteins DPAP A and Kex2p. Grd19p consists of the PX domain with 30 extra residues at the N-terminal and is homologous to the functionally characterized human sorting nexin protein SNX3. We determined the 2.0 A crystal structure of Grd19p in the free form and in complex with d-myo-phosphatidylinositol 3-phosphate (diC4PtdIns(3)P), representing the first case of both free and ligand-bound conformations of the same PX module. The ligand occupies a well defined positively charged binding pocket at the interface between the beta-sheet and alpha-helical parts of the molecule. The structure of the free and bound protein are globally similar but show some significant differences in a region containing a polyproline peptide and a putative membrane attachment site.

Seven in absentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the beta-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIP in vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP.