Polybromo-1-bromodomains bind histone H3 at specific acetyl-lysine positions

The human polybromo-1 protein is thought to localize the Polybromo, BRG1-associated factors chromatin-remodeling complex to kinetochores during mitosis via direct interaction of its six tandem bromodomains with acetylated nucleosomes. Bromodomains are acetyl-lysine binding modules roughly 100 amino acids in length originally found in chromatin associated proteins. Previous studies verified acetyl-histone binding by each bromodomain, but site-specificity, a central tenet of the histone code hypothesis, was not examined. Here, the acetylation site-dependence of bromodomain-histone interactions was examined using steady-state fluorescence anisotropy. Results indicate that single bromodomains bind specific acetyl-lysine sites within the histone tail with sub-micromolar affinity. Identification of duplicate target sites suggests that native Pb1 interacts with both copies of histone H3 upon nucleosome assembly. Quantitative analysis of single bromodomain-histone interactions can be used to develop hypotheses regarding the histone acetylation pattern that acts as the binding target of the native polybromo-1 protein.     

Structure of the yeast Hst2 protein deacetylase in ternary complex with 2'-O-acetyl ADP ribose and histone peptide

Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide. The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies. Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues.     

An autonomously folding beta-hairpin derived from the human YAP65 WW domain: attempts to define a minimum ligand-binding motif

WW domains are broadly distributed among natural proteins; these modules play a role in bringing specific proteins together. The ligands recognized by WW domains are short segments rich in proline residues. We have tried to identify the minimum substructure within a WW domain that is required for ligand binding. WW domains typically comprise ca. 40 residues and fold to a three-stranded beta-sheet. Structural data for several WW domain/ligand complexes suggest that most or all of the intermolecular contacts involve beta-strands 2 and 3. We have developed a 16-residue peptide that folds to a beta-hairpin conformation that appears to mimic beta-strands 2 and 3 of the human YAP65 WW domain, but this peptide does not bind to known ligands. Thus, the minimum binding domain is larger than the latter two strands of the WW domain beta-sheet.     

Structure and substrate binding properties of cobB, a Sir2 homolog protein deacetylase from Escherichia coli

Sirtuins are NAD+-dependent protein deacetylase enzymes that are broadly conserved from bacteria to human, and have been implicated to play important roles in gene regulation, metabolism and longevity. cobB is a bacterial sirtuin that deacetylates acetyl-CoA synthetase (Acs) at an active site lysine to stimulate its enzymatic activity. Here, we report the structure of cobB bound to an acetyl-lysine containing non-cognate histone H4 substrate. A comparison with the previously reported archaeal and eukaryotic sirtuin structures reveals the greatest variability in a small zinc-binding domain implicated to play a particularly important role in substrate-specific binding by the sirtuin proteins. Comparison of the cobB/histone H4 complex with other sirtuin proteins in complex with acetyl-lysine containing substrates, further suggests that contacts to the acetyl-lysine side-chain and beta-sheet interactions with residues directly C-terminal to the acetyl-lysine represent conserved features of sirtuin-substrate recognition. Isothermal titration calorimetry studies were used to compare the affinity of cobB for a variety of cognate and non-cognate acetyl-lysine-bearing peptides revealing an exothermic reaction with relatively little discrimination between substrates. In contrast, similar studies employing intact acetylated Acs protein as a substrate reveal a binding reaction that is endothermic, suggesting that cobB recognition of substrate involves a burial of hydrophobic surface and/or structural rearrangement involving substrate regions distal to the acetyl-lysine-binding site. Together, these studies suggest that substrate-specific binding by sirtuin proteins involves contributions from the zinc-binding domain of the enzyme and substrate regions distal to the acetyl-lysine-binding site.     

Ubiquitin binds to and regulates a subset of SH3 domains

SH3 domains are modules of 50-70 amino acids that promote interactions among proteins, often participating in the assembly of large dynamic complexes. These domains bind to peptide ligands, which usually contain a core Pro-X-X-Pro (PXXP) sequence. Here we identify a class of SH3 domains that bind to ubiquitin. The yeast endocytic protein Sla1, as well as the mammalian proteins CIN85 and amphiphysin, carry ubiquitin-binding SH3 domains. Ubiquitin and peptide ligands bind to the same hydrophobic groove on the SH3 domain surface, and ubiquitin and a PXXP-containing protein fragment compete for binding to SH3 domains. We conclude that a subset of SH3 domains constitutes a distinct type of ubiquitin-binding domain and that ubiquitin binding can negatively regulate interaction of SH3 domains with canonical proline-rich ligands.     

The CCN family of proteins: structure-function relationships

The CCN proteins are key signalling and regulatory molecules involved in many vital biological functions, including cell proliferation, angiogenesis, tumourigenesis and wound healing. How these proteins influence such a range of functions remains incompletely understood but is probably related to their discrete modular nature and a complex array of intra- and inter-molecular interactions with a variety of regulatory proteins and ligands. Although certain aspects of their biology can be attributed to the four individual modules that constitute the CCN proteins, recent results suggest that some of their biological functions require cooperation between modules. Indeed, the modular structure of CCN proteins provides important insight into their structure-function relationships.     

Improved methods for targeting epigenetic reader domains of acetylated and methylated lysine

Responsible for interpreting histone post-translational modifications, epigenetic reader proteins have emerged as novel therapeutic targets for a wide range of diseases. Chemical probes have been critical in enabling target validation studies and have led to translational advances in cancer and inflammation-related pathologies. Here, we present the most recently reported probes of reader proteins that recognize acylated and methylated lysine. We will discuss challenges associated with achieving potent antagonism of reader domains and review ongoing efforts to overcome these hurdles, focusing on targeting strategies including the use of peptidomimetic ligands, allosteric modulators, and protein degraders.     

Structure and function of laminin LG modules.

Laminin G domain-like (LG) modules of approximately 180-200 residues are found in a number of extracellular and receptor proteins and often are present in tandem arrays. LG modules are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. The recently determined crystal structures of LG modules of the laminin alpha2 chain reveal a compact beta sandwich fold and identify a novel calcium binding site. Binding epitopes for heparin, sulfatides and alpha-dystroglycan have been mapped by site-directed mutagenesis and show considerable overlap. The epitopes are located in surface loops around the calcium site, which in other proteins (agrin, neurexins) are modified by alternative splicing. Efficient ligand binding often requires LG modules to be present in tandem. The close proximity of the N- and C-termini in the LG module, as well as a unique link region between laminin LG3 and LG4, impose certain constraints on the arrangement of LG tandems. Further modifications may be introduced by proteolytic processing of laminin G domains, which is known to occur in the alpha2, alpha3 and alpha4 chains.     

Bromodomains识别并结合配体的机理研究

关于酵母重组蛋白内的Bromodomain识别乙酰化赖氨酸的研究近年来受到广泛的关注,但是其识别配体并与之相紧密结合的机理有待进一步的研究。本文采用2015年开发的结合位点拓扑学方法(FCTM)和分子动力学模拟的方式对Bromodomains识别并结合配体的机理进行了充分研究,其中分子动力学模拟时间达24 ns。通过FCTM方法发现结合位点的几何结构具有高度的凹性,且其alphaspace达到了131。分子动力学模拟的结果显示:在模拟的过程中结合位点表面的脯氨酸(Pro66)始终对配体保持着强的分子间相互作用,同时pocket内的水分子分布对配体的氢键网络也一直存在影响。以上结果表明Bromodomains识别并结合配体有两个重要因素:蛋白结构域自身的几何结构和配体受到来自于结合位点表面的氨基酸分子相互作用和pocket内水分子的氢键网络作用。     

Acetyl-lysine analog peptides as mechanistic probes of protein deacetylases

Class III histone deacetylases (Sir2 or sirtuins) catalyze the NAD+-dependent conversion of acetyl-lysine residues to nicotinamide, 2'-O-acetyl-ADP-ribose (OAADPr), and deacetylated lysine. Class I and II HDACs utilize a different deacetylation mechanism, utilizing an active site zinc to direct hydrolysis of acetyl-lysine residues to lysine and acetate. Here, using ten acetyl-lysine analog peptides, we have probed the substrate binding pockets of sirtuins and investigated the catalytic differences among sirtuins and class I and II deacetylases. For the sirtuin Hst2, acetyl-lysine analog peptide binding correlated with the hydrophobic substituent parameter pi with a slope of -0.35 from a plot of log Kd versus pi. Interestingly, propionyl- and butyryl-lysine peptides were found to bind tighter to Hst2 compared with acetyl-lysine peptide and showed measurable rates of catalysis with Hst2, Sirt1, Sirt2, and Sirt3, suggesting propionyl- and butyryl-lysine proteins may be sirtuin substrates in vivo. Unique among the acetyl-lysine analog peptides examined, homocitrulline peptide produced ADP-ribose instead of the corresponding OAADPr analog. The electron-withdrawing nature of each acetyl analog had a profound impact on the deacylation rate between deacetylase classes. The rate of catalysis with the acetyl-lysine analog peptides varied over five orders of magnitude with the class III deacetylase Hst2, revealing a linear free energy relationship with a slope of -1.57 when plotted versus the Taft constant, sigma*. HDAC8, a class I deacetylase, displayed the opposite trend with a slope of +0.79. These results are applicable toward the development of selective substrates and other mechanistic probes of protein deacetylases.     

On the Classification and Evolution of Protein Modules

Our efforts to classify the functional units of many proteins, the modules, are reviewed. The data from the sequencing projects for various model organisms are extremely helpful in deducing the evolution of proteins and modules. For example, a dramatic increase of modular proteins can be observed from yeast to C. elegans in accordance with new protein functions that had to be introduced in multicellular organisms. Our sequence characterization of modules relies on sensitive similarity search algorithms and the collection of multiple sequence alignments for each module. To trace the evolution of modules and to further automate the classification, we have developed a sequence and a module alerting system that checks newly arriving sequence data for the presence of already classified modules. Using these systems, we were able to identify an unexpected similarity between extracellular C1Q modules with bacterial proteins.     

Expression, purification and characterization of individual bromodomains from human Polybromo-1

Computational analysis reveals six tandem bromodomains within the amino-terminal region of the human Polybromo-1 protein, a required subunit of the Polybromo, BRG1-associated factors chromatin remodeling complex. Bromodomains are acetyl-lysine binding modules found in many chromatin binding proteins and histone acetyltransferases. Recent in vivo studies suggest that bromodomains can both discriminate the presence of an acetyl group on a lysine side chain and locate the acetyl-lysine within a histone protein. Together, this implies that multiple bromodomains may be able to function cooperatively and recognize a specific acetylation pattern to localize remodeling complexes to specific chromatin sites. Here, the cloning, expression and bioactivity of recombinant bromodomains from the human Polybromo-1 protein is described. Individual bromodomains from Polybromo-1 were cloned from human cDNA into a pET30b expression vector enabling effective one-step purification by affinity chromatography. Due to complications, including the high number of rare codons found in the coding regions and the tendency of individually expressed domains to aggregate and misfold, bacterial expression was only achieved using a cell strain containing rare eukaryotic tRNAs. Fluorescence-based bioactivity assays were performed to determine if native binding features were retained. The present cloning, expression, and purification procedure enabled the preparation of large quantity and high yields of biologically active recombinant bromodomains from human Polybromo-1 for in vitro structure and function studies. This is the first report of recombinant active form of bromodomains obtained from PB1.     

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.     

The dual-function disabled-1 PTB domain exhibits site independence in binding phosphoinositide and peptide ligands

While typical intracellular protein modules have only one ligand-binding site, there are rare examples of single modules that bind two different ligands at distinct binding sites. Here we present a detailed mutational and energetic analysis of one such domain, the phosphotyrosine binding (PTB) domain of Disabled-1 (Dab1), which binds to both peptide and phosphoinositide (PI) ligands simultaneously at structurally distinct binding sites. Through the techniques of isothermal titration calorimetry (ITC), analysis of Dab1 PTB domain mutants, and nuclear magnetic resonance (NMR), we have evaluated the characteristics of binding of the Dab1 PTB domain to various peptide and PI ligands. These studies reveal that the presence of saturating concentrations of one ligand has little effect on the binding constant for a second ligand at the other site. In addition, proteins with single-point mutations in the peptide-binding site retain native affinity for PI ligands, while proteins with mutations that prevent PI binding retain native affinity for peptide. NMR titrations show that the final structure of the ternary complex is the same independent of the order of addition of the two ligands. Together, these studies show that binding of peptide and PI ligands is energetically independent and noncooperative.