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The E3 ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) plays a critical role in regulating the ubiquitin-dependent degradation of misfolded proteins. CHIP mediates the ubiquitination of the α-amino-terminus of substrates with the E2 Ube2w and facilitates the ubiquitination of lysine residues with the E2 UbcH5. While it is known that Ube2w directly interacts with the disordered regions at the N-terminus of its substrates, it is unclear how CHIP and UbcH5 mediate substrate lysine selection. Here, we have decoupled the contributions of the E2, UbcH5, and the E3, CHIP, in ubiquitin transfer. We show that UbcH5 selects substrate lysine residues independent of CHIP, and that CHIP participates in lysine selection by fine-tuning the subset of substrate lysines that are ubiquitinated. We also identify lysine 128 near the C-terminus of UbcH5 as a critical residue for the efficient ubiquitin transfer by UbcH5 in both the presence and absence of CHIP. Together, these data demonstrate an important role of the UbcH5/substrate interactions in mediating the efficient ubiquitin transfer by the CHIP/UbcH5 complex.

This is a short paper on new ways to think about the structure and function of small heat shock proteins (sHSPs), perhaps the most enigmatic family among protein chaperones. The goal is to incorporate new observations regarding the disordered regions of small heat shock proteins (sHSPs) into the large body of structural information on the conserved structural alpha-crystallin domains (ACD) that define the sHSP family. Disordered regions (N-terminal region and C-terminal region or NTR and CTR, respectively) represent over 50% of the sHSP sequence space in the human genome and are refractory to traditional structural biology approaches, posing a roadblock on the path towards a mechanistic understanding of how sHSPs function. A model in which an ACD dimer serves as a template that presents three grooves into which other proteins or other segments of sHSPs can bind is presented. Short segments within the disordered regions are observed to bind into the ACD grooves. There are more binding segments than there are grooves, and each binding event is weak and transient, creating a dynamic equilibrium of tethered and untethered disordered regions. The ability of an NTR to be in dynamic equilibrium between tethered/sequestered and untethered states suggests several mechanistic alternatives that need not be mutually exclusive. New ways of thinking about (and approaching) the intrinsic properties of sHSPs may finally allow the veil of enigma to be removed from sHSPs.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an ‘inactive’ low-affinity state (LAS) to an ‘active’ high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH’s ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function., Author summary To bind their ligands, allosteric proteins shift between ‘inactive’ and ‘active’ states, but molecular details of the conformational changes during the transition are often unclear. We describe a monoclonal antibody against the mannose-specific bacterial adhesin, FimH, that blocks the conformational transition in both directions. The antibody-trapped LAS and HAS conformations of FimH are unable to mediate bacterial adhesion under dynamic shear conditions. We propose that the conformational trapping involves a steric block of the core-to-surface switching of certain residues which is critical for the allosteric transitions. Furthermore, we demonstrate that the allosteric switches are structurally and functionally conserved across a broad spectrum of bacterial fimbrial adhesins.

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a “fuzzy” protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein–protein interaction., The intrinsically disordered acidic activation domain (AD) of the yeast transcription factor Gal4 acts through binding to the Med15 subunit of the Mediator complex. Here, the authors show that Gal4 interacts with Med15 through an identical fuzzy binding mechanism as Gcn4 AD, which has a different sequence, revealing a common sequence-independent mechanism for AD-Mediator binding. In contrast, Gal4 AD binds to the Gal80 repressor as a structured polypeptide, which strongly suggests that the structured binding partner dictates the type of protein–protein interaction for an intrinsically disordered protein.

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme’s true physiological substrate., The Legionella pneumophila effector MavC inhibits the human ubiquitin-conjugating enzyme Ube2N. Here, the authors combine NMR, X-ray crystallography and biochemical assays and show that MavC catalyses the intramolecular transglutaminase reaction between the Ube2N and Ub subunits of the Ube2N∼Ub conjugate and present the substrate- and product-bound MavC crystal structures.

Poly(ADP‐ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase‐1 and ‐2; a.k.a. PARP‐5a and ‐5b) is involved in mitosis, telomere length regulation, GLUT‐4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase‐binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C‐terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate‐binding region of TNKS, forming a robust one‐to‐one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide‐binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase‐binding proteins including those of IF4A1 and NELFE.

Protein evolution is driven by the sum of different physiochemical and genetic processes that usually results in strong purifying selection to maintain biochemical functions. However, proteins that are part of systems under arms race dynamics often evolve at unparalleled rates that can produce atypical biochemical properties. In the marine mollusk abalone, lysin and VERL are a pair of rapidly coevolving proteins that are essential for species-specific interactions between sperm and egg. Despite extensive biochemical characterization of lysin, including crystal structures of multiple orthologs, it was unclear how sites under positive selection may facilitate recognition of VERL. Using a combination of targeted mutagenesis and multidimensional NMR, we present a high-definition solution structure of sperm lysin from red abalone (Haliotis rufescens). Unapparent from the crystallography data, multiple NMR-based analyses conducted in solution reveal clustering of the N-and C-termini to form a nexus of 13 positively selected sites that constitute a VERL binding interface. Evolutionary rate was found to be a significant predictor of backbone flexibility, which may be critical for lysin bioactivity and / or accelerated evolution. These flexible, rapidly evolving segments that constitute the VERL binding interface were also the most distorted regions of the crystal structure relative to what was observed in solution. While lysin has been the subject of extensive biochemical and evolutionary analyses for more than 30 years, this study highlights the enhanced insights gained from applying NMR approaches to rapidly evolving proteins.SignificanceThe fertilization of eggs by sperm is a critical biological process for nearly all sexually reproducing organisms to propagate their genetic information. Despite the importance of fertilization, the molecules that mediate egg-sperm interactions have been characterized for only a few species, and the biochemical mechanisms underlying these interactions are even less well understood. In the marine mollusk abalone, sperm lysin interacts with egg VERL in a species-specific manner to facilitate fertilization. In this report, we characterized the solution structure and molecular evolution of sperm lysin from red abalone (Haliotis rufescens), and identified the VERL binding interface as well as important lysin dimer interactions that have emerged as part of an incessant sexual arms race.

Small heat shock proteins (sHPSs) are nature’s “first responders” to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of “quasi-ordered” NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.

RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.

Small heat shock proteins (sHSP) are a class of ATP-independent protein chaperones found throughout nature. They share a common ability to maintain partly unfolded proteins in soluble states under cellular stress conditions. All sHSPs contain a central domain called the α-crystallin domain (ACD); the domain is found in all sHSPs and in no other proteins and therefore defines the family. Though most sHSPs form large, often polydisperse oligomers from varying numbers of subunits, the ACD is both necessary and sufficient for formation of a dimer, the fundamental building block for oligomers. HSPB1 (also known as Hsp27) is unique among the ten human sHSPs because it contains a Cys residue in its dimer interface. HSPB1 is highly expressed under conditions of oxidative stress and is proposed to serve as a redox-sensitive chaperone. HSPB1 residue Cys137 has been proposed to modulate function by existing in either its oxidized (disulfide) or reduced (thiol) form (Chalova et al 2014). Here we report the solution-state NMR structure of oxidized HSPB1-ACD and compare it to a previously determined crystal structure of the reduced state. Formation of the disulfide-bond across the dimer interface yields a locked dimer structure with increased accessible hydrophobic surface. In the context of full-length HSPB1 oligomers, oxidation of Cys137 is associated with enhanced ability to bind the hydrophobic dye, 8-Anilinonapthalene-1-sulfonic-acid, implying an increased ability to interact with client proteins under oxidative stress.

Small heat shock proteins (sHSPs) make up a class of molecular chaperones broadly observed across organisms. Many sHSPs form large oligomers that undergo dynamic subunit exchange that is thought to play a role in chaperone function. Though remarkably heterogeneous, sHSP oligomers share three types of intermolecular interactions that involve all three defined regions of a sHSP: the N-terminal region (NTR), the conserved α-crystallin domain (ACD), and a C-terminal region (CTR). Here we define the structural interactions involved in incorporation of a subunit into a sHSP oligomer. We demonstrate that a minimal ACD dimer of the human sHSP, HSPB5, interacts with an HSPB5 oligomer through two types of interactions: (1) interactions with CTRs in the oligomer and (2) via exchange into and out of the dimer interface composed of two ACDs. Unexpectedly, although dimers are thought to be the fundamental building block for sHSP oligomers, our results clearly indicate that subunit exchange into and out of oligomers occurs via monomers. Using structure-based mutants, we show that incorporation of a subunit into an oligomer is predicated on recruitment of the subunit via its interaction with CTRs on an oligomer. Both the rate and extent of subunit incorporation depend on the accessibility of CTRs within an HSPB5 oligomer. We show that this mechanism also applies to formation of heterooligomeric sHSP species composed of HSPB5 and HSPB6 and is likely general among sHSPs. Finally, our observations highlight the importance of NTRs in the thermodynamic stability of sHSP oligomers.

The holdase activity and oligomeric propensity of human small heat shock proteins (sHSPs) are regulated by environmental factors. However, atomic-level details are lacking for the mechanisms by which stressors alter sHSP responses. We previously demonstrated that regulation of HSPB5 is mediated by a single conserved histidine over a physiologically relevant pH range of 6.5–7.5. Here, we demonstrate that HSPB1 responds to pH via a similar mechanism through pH-dependent structural changes that are induced via protonation of the structurally analogous histidine. Results presented here show that acquisition of a positive charge, either by protonation of His124 or its substitution by lysine, reduces the stability of the dimer interface of the α-crystallin domain, increases oligomeric size, and modestly increases chaperone activity. Our results suggest a conserved mechanism of pH-dependent structural regulation among the human sHSPs that possess the conserved histidine, although the functional consequences of the structural modulations vary for different sHSPs.

Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.

Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7, 8, 9). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU). Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso-ADP-ribose (iso-ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso-ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

RING finger domain and RING finger-like ubiquitin ligases (E3s), such as U-box proteins, constitute the vast majority of known E3s. RING-type E3s function together with ubiquitin-conjugating enzymes (E2s) to mediate ubiquitination and are implicated in numerous cellular processes. In part because of their importance in human physiology and disease, these proteins and their cellular functions represent an intense area of study. Here we review recent advances in RING-type E3 recognition of substrates, their cellular regulation, and their varied architecture. Additionally, recent structural insights into RING-type E3 function, with a focus on important interactions with E2s and ubiquitin, are reviewed. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86–130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.

RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2∼ubiquitin (E2∼Ub) via their RINGs but canonical RING E3s promote closed E2∼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2∼Ub to favor Ub transfer to the E3 active site. This different RING/E2∼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2∼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.

The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a "fuzzy" complex, where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.

SummaryThe Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the β sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, β sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ∼ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ∼ Ub n ). Rapid generation of UbcH5 ∼ Ub n may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.

Substantial evidence has accumulated indicating a significant role for oligomerization in the function of E3 ubiquitin ligases. Among the many characterized E3 ligases, the yeast U-box protein Ufd2 and its mammalian homolog E4B appear to be unique in functioning as monomers. An E4B U-box domain construct (E4BU) has been sub-cloned, over-expressed in E. Coli and purified, which enabled determination of a high resolution NMR solution structure and detailed biophysical analysis. E4BU is a stable monomeric protein that folds into the same structure observed for other structurally characterized U-box domains, all of which are homodimers. Multiple sequence alignment combined with comparative structural analysis reveals substitutions in the sequence that inhibit dimerization. The interaction between E4BU and the E2 conjugating enzyme UbcH5c has been mapped using NMR and this data has been used to generate a structural model for the complex. The E2 binding site is found to be similar to that observed for dimeric U-box and RING domain E3 ligases. Despite the inability to dimerize, E4BU was found to be active in a standard autoubiquitination assay. The structure of E4BU and its ability to function as a monomer are discussed in light of the ubiquitous observation of U-box and RING domain oligomerization.

GAF domains regulate the catalytic activity of certain vertebrate cyclic nucleotide phosphodiesterases (PDEs) by allosteric, noncatalytic binding of cyclic nucleotides. GAF domains arranged in tandem are found in PDE2, −5, −6, −10, and −11, all of which regulate the cellular concentrations of the second messengers cAMP and/or cGMP. Nucleotide binding to GAF domains affects the overall conformation and the catalytic activity of full-length PDEs. The cyclic nucleotide-bound GAF domains from PDE2, −5, −6, and −10 all adopt a conserved fold but show subtle differences within the binding pocket architecture that account for a large range of nucleotide affinities and selectivity. NMR data and details from the structure of full-length nucleotide-free PDE2A reveal the dynamic nature and magnitude of the conformational change that accompanies nucleotide binding. The discussed GAF domain structures further reveal differences in dimerization properties and highlight the structural diversity within GAF domain-containing PDEs.

Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.

Anomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine-containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (delta delta G(folding) = 0.46 +/- 0.15 kcal mol-1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche- conformer in wild-type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main-chain amide proteins in addition to its interaction with the side chain of Ser 31 in the wild-type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side-chain hydrophobicity and conformational entropy between the two proteins are accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical shift mapping is becoming a popular method for studying protein-protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N)-HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non-specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.

Summary The PhoQ sensor kinase is essential for Salmonella typhimurium virulence for animals, and a homologue exists in the environmental organism and opportunistic pathogen Pseudomonas aeruginosa. S. typhimurium PhoQ (ST-PhoQ) is repressed by millimolar concentrations of divalent cations and activated by antimicrobial peptides and at acidic pH. ST-PhoQ has a periplasmic Per-ARNT-Sim domain, a fold commonly employed for ligand binding. However, substrate binding is instead accomplished by an acidic patch in the periplasmic domain that interacts with the inner membrane through divalent cation bridges. The DNA sequence encoding this acidic patch is absent from Pseudomonas phoQ (PA-PhoQ). Here, we demonstrate that PA-PhoQ binds and is repressed by divalent cations, and can functionally complement a S. typhimurium phoQ-null mutant. Mutational analysis and NMR spectroscopy of the periplasmic domains of ST-PhoQ and PA-PhoQ indicate distinct mechanisms of binding divalent cation. The data are consistent with PA-PhoQ binding metal in a specific ligand-binding pocket. PA-PhoQ was partially activated by acidic pH but not by antimicrobial peptides. S. typhimurium expressing PA-PhoQ protein were attenuated for virulence in a mouse model, suggesting that the ability of Salmonella to sense host environments via antimicrobial peptides and acidic pH is an important contribution to pathogenesis.

Since its discovery as a post-translational signal for protein degradation, our understanding of ubiquitin (Ub) has vastly evolved. Today, we recognize that the role of Ub signaling is expansive and encompasses diverse processes including cell division, the DNA damage response, cellular immune signaling, and even organismal development. With such a wide range of functions comes a wide range of regulatory mechanisms that control the activity of the ubiquitylation machinery. Ub attachment to substrates occurs through the sequential action of three classes of enzymes, E1s, E2s, and E3s. In humans, there are 2 E1s, ∼ 35 E2s, and hundreds of E3s that work to attach Ub to thousands of cellular substrates. Regulation of ubiquitylation can occur at each stage of the stepwise Ub transfer process, and substrates can also impact their own modification. Recent studies have revealed elegant mechanisms that have evolved to control the activity of the enzymes involved. In this minireview, we highlight recent discoveries that define some of the various mechanisms by which the activities of E3-Ub ligases are regulated.

Small heat shock proteins (sHSPs) are essential ‘holdase’ chaperones that form large assemblies and respond dynamically to pH and temperature stresses to protect client proteins from aggregation. While the alpha-crystallin domain (ACD) dimer of sHSPs is the universal building block, how the ACD transmits structural changes in response to stress to promote holdase activity is unknown. We found that the dimer interface of HSPB5 is destabilized over physiological pHs and a conserved histidine (His-104) controls interface stability and oligomer structure in response to acidosis. Destabilization by pH or His-104 mutation shifts the ACD from dimer to monomer but also results in a large expansion of HSPB5 oligomer states. Remarkably, His-104 mutant-destabilized oligomers are efficient holdases that reorganize into structurally distinct client–bound complexes. Our data support a model for sHSP function wherein cell stress triggers small perturbations that alter the ACD building blocks to unleash a cryptic mode of chaperone action. DOI: http://dx.doi.org/10.7554/eLife.07304.001, eLife digest Proteins are composed of one or more long chain-like molecules that must fold into complex three-dimensional shapes in order to work properly. Incorrectly folded proteins cannot function and often aggregate into toxic states that are associated with a number of neurological diseases including Alzheimer's, Huntington's, and Parkinson's. Elevated temperatures, increased acidity, and other stressful conditions in the cell can hinder the folding process and may cause existing proteins to unfold and aggregate. However, when cells experience these stresses, certain proteins—known as small heat shock proteins (or sHSPs for short)—act as ‘holdase chaperones’ to protect cells from protein misfolding. HSPB5 is one such chaperone that binds to and stabilizes other proteins (called ‘clients’) to prevent their aggregation. The core structure of HSPB5 and other similar chaperone proteins is well known. But, it is not clear how chaperones sense stressful conditions and respond to increase their activity to help stabilize client proteins. Now, Rajagopal et al. have identified a single amino acid in HSPB5 that is sensitive to pH changes. When the environment inside a cell becomes more acidic, this amino acid (a histidine) triggers changes in HSPB5's structure that enhance the chaperone's activity. This histidine was then replaced with another amino acid in an attempt to lock HSPB5 into a low-pH state that mimics an active HSPB5 chaperone inside a stressed cell. Further experiments revealed that this mutant HSPB5 is a super-active holdase chaperone, and that it dramatically changes its structure to bind to a client protein in the holdase state. From this, Rajagopal et al. propose a model to explain how cellular stress triggers small changes in HSPB5 that propagate through the chaperone in a response mechanism that increases its activity. Future studies will investigate whether inherited mutations in HSPB5 and other similar chaperones—which are associated with cardiac, muscle, and nerve disorders—exert their effect by disrupting this response mechanism. DOI: http://dx.doi.org/10.7554/eLife.07304.002

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQW104C-A128C is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQW104C-A128C Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence. DOI: http://dx.doi.org/10.7554/eLife.06792.001, eLife digest Salmonella bacteria cause illnesses in humans, such as food poisoning and typhoid fever. In response to a Salmonella infection, immune cells known as macrophages detect and engulf the bacteria. The conditions inside the macrophage (which include an acidic pH and high levels of antimicrobial molecules) can destroy some bacteria. However, Salmonella bacteria (which are also called salmonellae) can sense and counteract these hostile conditions; this allows them to remodel their surface to survive and reproduce inside macrophages and continue to cause disease. A protein known as PhoQ, which is found on the surface of Salmonella bacteria, is a sensor that detects when the bacterium is inside a macrophage and so needs to boost its defenses. The PhoQ sensor is able to respond to acidity, the absence of divalent cations—such as magnesium and calcium ions—and certain antimicrobial peptide molecules. These conditions and components are used inside macrophages to try and kill the bacteria, but it was not known which of these signals PhoQ actually senses during an infection. Hicks et al. established how the sensor region of PhoQ changes when it is exposed to acid. This knowledge enabled variants of this protein to be constructed that do not respond when exposed to acidic conditions or low levels of divalent cations. Salmonellae that have these modified PhoQ sensors were still able to infect macrophages and cause disease in mice. These findings suggest that antimicrobial peptide sensing alone is sufficient to trigger the bacteria's defenses inside host organisms. Understanding how salmonellae detect antimicrobial factors could help with the development of new treatments for the diseases caused by these bacteria. Furthermore, the new tools developed by Hicks et al. could be applied to other systems to characterize how bacteria interact with their host environment during infection. DOI: http://dx.doi.org/10.7554/eLife.06792.002

Muscarinic acetylcholine receptors comprise a family of G-protein-coupled receptors that display differential localization in polarized epithelial cells. We identify a seven-residue sequence, Ala(275)-Val(281), in the third intracellular loop of the M(3) muscarinic receptor that mediates dominant, position-independent basolateral targeting in Madin-Darby canine kidney cells. Mutational analyses identify Glu(276), Phe(280), and Val(281) as critical residues within this sorting motif. Phe(280) and Val(281) comprise a novel dihydrophobic sorting signal as mutations of either residue singly or together with leucine do not disrupt basolateral targeting. Conversely, Glu(276) is required and cannot be substituted with alanine or aspartic acid. A 19-amino acid peptide representing the M(3) sorting signal and surrounding sequence was analyzed via two-dimensional nuclear magnetic resonance spectroscopy. Solution structures show that Glu(276) resides in a type IV beta-turn and the dihydrophobic sequence Phe(280)Val(281) adopts either a type I or IV beta-turn.

SummaryParkin is an E3 ubiquitin ligase, mutations in which are responsible for autosomal recessive juvenile parkinsonism. Recently, several structures of Parkin have been solved, revealing its serpentine shape and modes of auto-inhibition.

BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RING/RING heterodimer. The BRCA1/BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1/BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn 2+ -loops and central α-helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1/BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

The minimal DNA-binding domain of the yeast ADR1 transcription factor consists of two Cys2-His2 zinc fingers and an additional 20 residues N-terminal and proximal to the fingers. The accessory sequence likely plays a role in contacting DNA. Paramagnetic cobalt was incorporated into the fingers of an ADR1 DNA-binding construct (ADR1z) to serve as a probe of the proximity of the accessory sequence to the zinc fingers. NMR signals from the accessory region are not perturbed by cobalt incorporation. Previous studies showed that this region is random coil in the ADR1z construct in the absence of DNA; it does not adopt a fixed orientation with respect to the cobalt sites. In contrast, many residues of the accessory region are perturbed by cobalt in the DNA-bound form of the protein, suggesting this region becomes constrained. This observation agrees with previous results showing a disorder-to-order transition for the accessory region upon DNA binding. Furthermore, these results indicate that the accessory region lies close to the fingers in the protein-DNA complex. This region thus does not extend along the DNA away from the zinc fingers; it more likely binds the same stretch of DNA contacted by the zinc fingers. Comparison to the behavior of other zinc-finger proteins that utilize an accessory DNA-binding sequence suggested that the region of ADR1 proximal to the zinc fingers might form an alpha-helix. Analysis of sequential NOEs in the accessory region of DNA-bound ADR1z reveals no helical structure.

Post-translational modification of proteins with ubiquitin is mediated by dynamic multienzyme machinery (E1, E2, and E3). E3 ubiquitin ligases play a key role acting as both scaffolds to bring reactants together and activators to catalyze ubiquitin (Ub) transfer from E2~Ub conjugates to substrates. Our recent studies provided insights into the mechanism of the activation event; binding of an E3 to an E2~Ub conjugate was found to affect the motions of E2~Ub and allosterically stimulate Ub transfer. This proposed mechanism implies that the dynamics of the conjugate, which has been shown to occupy a wide range of E2~Ub orientations, will be altered significantly upon binding of E3. To directly assess the effect of E3 binding on E2~Ub dynamics, we undertook an in-depth comparative analysis of (15)N nuclear magnetic resonance relaxation of UbcH5c~Ub in the absence and presence of the E3 ligase, E4B. Challenges encountered in deciphering interdomain motions for this ternary complex are discussed along with the limitations of the current approaches. Notably, although a reduction in interdomain dynamics of UbcH5c~Ub is observed upon binding to E4B, Ub retains an extensive degree of flexibility. These results provide strong support for our dynamic model of a significant orientational bias of Ub toward a more closed conformation in the E3/E2~Ub complex.

Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.

We have measured deuterium/hydrogen fractionation in three histidine-containing proteins, ecHPr, ecHPr mutant S31A, and bsHPr, and in random coil peptides using NMR and mass spectrometry. The amide protons of unstructured peptides exhibit equilibrium enrichment for deuterium, in agreement with previous studies. Enrichment for both protium and deuterium was observed in both HPrs, with fractionation factors ranging from 0.63 to 1.41. Enrichment for protium was seen in alpha-helical secondary structure. 'Strong' HBs previously identified by mutagenesis and thermodynamic measurements are significantly enriched for protium. Sites of protium enrichment are conserved in a structural context across species lines, though ecHPr and bsHPr share only 30% sequence identity, suggesting that strong HBs are conserved and may play an important role in stabilizing the folded state.

The serine-phosphorylated form of histidine-containing protein (HPr), a component of the phosphoenolpyruvate:sugar phosphotransferase system from Bacillus subtilis, has been characterized by NMR spectroscopy and solvent denaturation studies. The results indicate that phosphorylation of Ser 46, the N-cap of alpha-helix-B, does not cause a conformational change but rather stabilizes the helix. Amide proton exchange rates in helix-B are decreased and phosphorylation stabilizes the protein to solvent and thermal denaturation, with a delta delta G of 0.7-0.8 kcal mol-1. A mutant in which Ser 46 is replaced by aspartic acid shows a similar stabilization, indicating that an electrostatic interaction between the negatively charged groups and the helix macrodipole contributes significantly to the stabilization.

GTP, as well as other nucleoside triphosphates, stimulates the activity of Escherichia coli adenylyl cyclase in permeable cells; the stimulatory effect is lost when the cells are disrupted by passage through a French pressure cell. These data suggested that the allosteric regulation by GTP of adenylyl cyclase activity requires an interaction of the enzyme with other protein factors. Strains deleted for genes encoding proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) failed to show an activity stimulation by GTP. With a view to localizing the site of interaction of GTP with the adenylyl cyclase complex, a variety of studies using purified PTS proteins were performed using the photoaffinity labeling reagent, 8-azidoGTP. These studies showed that 8-azidoGTP bound specifically to HPr. A species specificity study showed that the photoaffinity reagent labeled E. coli HPr but not HPr proteins from Mycoplasma capricolum or Bacillus subtilis. A variety of site-directed mutations of E. coli HPr were evaluated for interaction with GTP by photoaffinity labeling as well as by nuclear magnetic resonance; the results of these studies indicate that the lysine residues at positions 24 and 27, serine-46, the threonine at position 36, and the aspartate at position 69 are important for the binding of GTP to HPr. Molecular modeling has been used to formulate a model for the binding of GTP to HPr involving electrostatic interaction of the phosphate groups of the nucleotide with the side chains of lysine residues 27 and 45 and serine-43, interaction of the sugar with serine-46, and interaction of the base with lysine-24. From these data, it is hypothesized that the binding of GTP to HPr is required for the GTP-dependent stimulation of the activity of the adenylyl cyclase complex.

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes including protein quality control, DNA repair, endocytosis and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the Ub C-terminus and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly-linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent “backside” interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the “infinite spiral” helical arrangement of the sole previously reported structure of an oligomeric conjugate. Our structure also differs in intra-conjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intra-conjugate relative E2/Ub orientations. We delineate a common intra-conjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 ligase CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3 ligases, which have important consequences for the ubiquitination process.

Summary Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system involves a series of reactions in which phosphoprotein intermediates are formed. Histidine-containing protein (HPr) is phosphorylated on the N delta 1 position of the imidazole ring of His15 by enzyme I and acts as a phosphoryl donor to the sugar-specific enzymes IIA. The structure of phosphorylated HPr from Bacillus subtilis, primarily, and from Escherichia coli has been studied by nuclear magnetic resonance (NMR) spectroscopy. Phosphorylation of His15 results in large downfield shifts in amide proton and nitrogen resonances for residues 16 and 17 but results in only modest or no shifts in other backbone resonances. The exchange rates of these two amide groups are decreased more than 10-fold upon phosphorylation. Analysis of the coupling constants 3JNH alpha revealed no significant changes throughout the protein, indicating that backbone phi dihedral angles do not change detectably. 3J alpha beta and 3JN beta patterns determined from P.E.COSY and HNHB spectra, respectively, revealed a change in one side chain, that of conserved Arg17. Analysis of NOESY spectra revealed a limited number of changes in NOEs involving protons in Ser12, His15, Arg71, and Pro18 in B. subtilis HPr. The NMR results indicate that the Arg17 side chain becomes limited in its conformational range in the phosphorylated protein, taking on a conformation that points its guanidinium group toward the phosphoryl group on His15. In addition, the tautomeric and ionization states of His15 have been determined using 15N and 31P NMR. At neutral pH, the imidazole is predominantly in the protonated form and the phosphoryl group is in the dianionic form in P-His15. Altogether, the results indicate that phosphorylation of His15 yields only a local effect on the protein's structure. The data are consistent with a small change in the disposition of the histidine side chain, allowing phosphoryl group oxygens to serve as hydrogen bond acceptors for the amide protons of residues Ala16 and Arg17, which constitute the first two residues of an alpha-helix. Thus, similar to many proteins that bind phosphoryl moieties noncovalently, the phosphoryl group in P-His15-HPr is situated to allow for a favorable electrostatic interaction at the N-terminal end of an alpha-helix.

The solution structure for a mutant zinc finger peptide based on the sequence of the C-terminal ADR1 finger has been determined by two-dimensional NMR spectroscopy. The mutant peptide, called PAPA, has both proline residues from the wild-type sequence replaced with alanines. A nonessential cysteine was also replaced with alanine. The behavior of PAPA in solution implicates the prolines in the conformational heterogeneity reported earlier for the wild-type peptide [Xu, R. X., Horvath, S. J., & Klevit, R. E. (1991) Biochemistry 30, 3365-3371]. The solution structure of PAPA reveals several interesting features of the zinc finger motif. The residue immediately following the second cysteine ligand adopts a positive phi angle, which we propose is a common feature of this class of zinc fingers, regardless of whether this residue is a glycine. The NMR spectrum and resulting solution structure of PAPA suggest that a side-chain to side-chain hydrogen bond involving an arginine and an aspartic acid analogous to one observed in the Zif268 protein-DNA cocrystal structure exists in solution in the absence of DNA [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. A model for the interaction between the two ADR1 zinc fingers and their DNA binding sites was built by superpositioning the refined solution structures of PAPA and ADR1b onto the Zif268 structure. This model offers structural explanations for a variety of mutations to the ADR1 zinc finger domains that have been shown to affect DNA-binding affinity or specificity.

The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.

The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of approximately 121 degrees between the planes of the beta-sandwich formed by alpha-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry.

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.