High‐energy water sites determine peptide binding affinity and specificity of PDZ domains

PDZ domains have well known binding preferences for distinct C‐terminal peptide motifs. For most PDZ domains, these motifs are of the form [S/T]‐W‐[I/L/V]. Although the preference for S/T has been explained by a specific hydrogen bond interaction with a histidine in the PDZ domain and the (I/L/V) is buried in a hydrophobic pocket, the mechanism for Trp specificity at the second to last position has thus far remained unknown. Here, we apply a method to compute the free energies of explicit water molecules and predict that potency gained by Trp binding is due to a favorable release of high‐energy water molecules into bulk. The affinities of a series of peptides for both wild‐type and mutant forms of the PDZ domain of Erbin correlate very well with the computed free energy of binding of displaced waters, suggesting a direct relationship between water displacement and peptide affinity. Finally, we show a correlation between the magnitude of the displaced water free energy and the degree of Trp‐sensitivity among subtypes of the HTRA PDZ family, indicating a water‐mediated mechanism for specificity of peptide binding.     

Uncovering new aspects of protein interactions through analysis of specificity landscapes in peptide recognition domains

Protein interactions underlie all biological processes. An important class of protein interactions, often observed in signaling pathways, consists of peptide recognition domains binding short protein segments on the surface of their target proteins. Recent developments in experimental techniques have uncovered many such interactions and shed new lights on their specificity. To analyze these data, novel computational methods have been introduced that can accurately describe the specificity landscape of peptide recognition domains and predict new interactions. Combining large-scale analysis of binding specificity data with structure-based modeling can further reveal new biological insights into the molecular recognition events underlying signaling pathways.     

Sequence-specific dynamics modulate recognition specificity in WW domains

The current canon attributes the binding specificity of protein-recognition motifs to distinctive chemical moieties in their constituent amino acid sequences. However, we show for a WW domain that the sequence crucial for specificity is an intrinsically flexible loop that partially rigidifies upon ligand docking. A single-residue deletion in this loop simultaneously reduces loop flexibility and ligand binding affinity. These results suggest that sequences of recognition motifs may reflect natural selection of not only chemical properties but also dynamic modes that augment specificity.     

Role of SR protein modular domains in alternative splicing specificity in vivo

The SR proteins constitute a family of nuclear phosphoproteins which are required for constitutive splicing and also influence alternative splicing regulation. They have a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain, rich in arginine and serine residues. The functional role of the different domains of SR proteins in constitutive splicing activity has been extensively studied in vitro; however, their contribution to alternative splicing specificity in vivo has not been clearly established. We sought to address how the modular domains of SR proteins contribute to alternative splicing specificity. The activity of a series of chimeric proteins consisting of domain swaps between different SR proteins showed that splice site selection is determined by the nature of the RRMs and that RRM2 of SF2/ASF has a dominant role and can confer specificity to a heterologous protein. In contrast, the identity of the RS domain is not important, as the RS domains are functionally interchangeable. The contribution of the RRMs to alternative splicing specificity in vivo suggests that sequence-specific RNA binding by SR proteins is required for this activity.     

Can ungulates foraging in a multiple‐use landscape alter forest nitrogen budgets?

Ungulates alter nutrient dynamics within many of Earth's ecosystems. However, whether foraging ungulates transfer nutrients among ecosystems at a magnitude sufficient to alter nutrient budgets and associated ecological processes is unknown. I suggest that within eastern North American landscapes dominated by agriculture and forest, the juxtaposition of high nitrogen crops for foraging and forest used for diurnal concealment promotes a net transfer of nitrogen from cropland to forests by overabundant white-tailed deer ( Odocoileus virginianus ). To estimate the magnitude of this nutrient transfer I apply allometric relationships of deer nitrogen consumption and excretion to populations within a hypothetical landscape in which deer use forest and cropland in equal proportions daily and are not limited in habitat use by landscape spatial pattern. Results indicate a non-linear relationship between deer nitrogen deposition to forest and percent forest cover, with deposition rising toward estimated atmospheric nitrogen deposition when forest covers less than 40% of the landscape. This "spatial subsidy" of nitrogen to the forest-floor represents a previously unrecognized input to the forest nitrogen budget with equally unknown impacts on forest-floor ecosystem processes.     

The recognition of membrane‐bound PtdIns3P by PX domains

Phox‐homology (PX) domains target proteins to the organelles of the secretary and endocytic systems by binding to phosphatidylinositol phospholipids (PIPs). Among all the structures of PX domains that have been solved, only three have been solved in a complex with the main physiological ligand: PtdIns3P. In this work, molecular dynamic simulations have been used to explore the structure and dynamics of the p40^phox–PX domain and the SNX17–PX domain and their interaction with membrane‐bound PtdIns3P. In the simulations, both PX domains associated spontaneously with the membrane‐bound PtdIns3P and formed stable complexes. The interaction between the p40^phox–PX domain and PtdIns3P in the membrane was found to be similar to the crystal structure of the p40^phox–PX–PtdIns3P complex that is available. The interaction between the SNX17–PX domain and PtdIns3P was similar to that observed in the p40^phox–PX–PtdIns3P complex; however, some residues adopted different orientations. The simulations also showed that nonspecific interactions between the β1–β2 loop and the membrane play an important role in the interaction of membrane bound PtdIns3P and different PX domains. The behaviour of unbound PtdIns3P within a 2‐oleoyl‐1‐palmitoyl‐sn‐glycero‐3‐phosphocholine (POPC) membrane environment was also examined and compared to the available experimental data and simulation studies of related molecules. Proteins 2014; 82:2332–2342. © 2014 Wiley Periodicals, Inc.     

Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries

Signaling complexes usually involve multidomain proteins containing catalytic domains and peptide recognition modules (PRMs), which mediate protein-protein interactions and assemble complexes by binding to ligands containing a core sequence motif. Concomitant to large-scale physical interaction screening, considerable effort has been devoted toward the elucidation of consensus profiles for common PRMs. We describe herein a robust and proven protocol to generate consensus profiles for PRMs using phage-displayed peptide libraries. The initial phase of the protocol entails the cloning, expression and purification of PRMs as fusion proteins, in addition to the construction of highly diverse phage-displayed peptide libraries. The affinity selection process described thereafter enables a single researcher to efficiently probe the recognition profiles of numerous PRMs in a 1 week time period.     

Conserved signal peptide recognition systems across the prokaryotic domains

The twin-arginine translocation (Tat) pathway is a protein targeting system found in bacteria, archaea, and chloroplasts. Proteins are directed to the Tat translocase by N-terminal signal peptides containing SRRxFLK "twin-arginine" amino acid motifs. The key feature of the Tat system is its ability to transport fully folded proteins across ionically sealed membranes. For this reason the Tat pathway has evolved for the assembly of extracytoplasmic redox enzymes that must bind cofactors, and so fold, prior to export. It is important that only cofactor-loaded, folded precursors are presented for export, and cellular processes have been unearthed that regulate signal peptide activity. One mechanism, termed "Tat proofreading", involves specific signal peptide binding proteins or chaperones. The archetypal Tat proofreading chaperones belong to the TorD family, which are dedicated to the assembly of molybdenum-dependent redox enzymes in bacteria. Here, a gene cluster was identified in the archaeon Archaeoglobus fulgidus that is predicted to encode a putative molybdenum-dependent tetrathionate reductase. The gene cluster also encodes a TorD family chaperone (AF0160 or TtrD) and in this work TtrD is shown to bind specifically to the Tat signal peptide of the TtrA subunit of the tetrathionate reductase. In addition, the 3D crystal structure of TtrD is presented at 1.35 ? resolution and a nine-residue binding epitope for TtrD is identified within the TtrA signal peptide close to the twin-arginine targeting motif. This work suggests that archaea may employ a chaperone-dependent Tat proofreading system that is similar to that utilized by bacteria.     

In vitro evolution of recognition specificity mediated by SH3 domains reveals target recognition rules

We have designed a repertoire of 10(7) different SH3 domains by grafting the residues that are represented in the binding surfaces of natural SH3 domains onto the scaffold of the human Abl-SH3 domain. This phage-displayed library was screened by affinity selection for SH3 domains that bind to the synthetic peptides, APTYPPPLPP and LSSRPLPTLPSP, which are peptide ligands for the human Abl or Src SH3 domains, respectively. By characterizing the isolates, we have observed that as few as two or three amino acid substitutions lead to dramatic changes in recognition specificity. We propose that the ability to shift recognition specificity with a small number of amino acid replacements is an important evolutionary characteristic of protein binding modules. Furthermore, we have used the information obtained by these in vitro evolution experiments to generate a scoring matrix that evaluates the probability that any SH3 domain binds to the peptide ligands for the Abl and Src SH3 domains. A table of predictions for the 28 SH3 domains of baker's yeast is presented.     

Comprehensive modeling and functional analysis of Toll‐like receptor ligand‐recognition domains

Toll‐like receptors (TLRs) are innate immune pattern‐recognition receptors endowed with the capacity to detect microbial pathogens based on pathogen‐associated molecular patterns. The understanding of the molecular principles of ligand recognition by TLRs has been greatly accelerated by recent structural information, in particular the crystal structures of leucine‐rich repeat‐containing ectodomains of TLR2, 3, and 4 in complex with their cognate ligands. Unfortunately, for other family members such as TLR7, 8, and 9, no experimental structural information is currently available. Methods such as X‐ray crystallography or nuclear magnetic resonance are not applicable to all proteins. Homology modeling in combination with molecular dynamics may provide a straightforward yet powerful alternative to obtain structural information in the absence of experimental (structural) data, provided that the generated three‐dimensional models adequately approximate what is found in nature. Here, we report the development of modeling procedures tailored to the structural analysis of the extracellular domains of TLRs. We comprehensively compared secondary structure, torsion angles, accessibility for glycosylation, surface charge, and solvent accessibility between published crystal structures and independently built TLR2, 3, and 4 homology models. Finding that models and crystal structures were in good agreement, we extended our modeling approach to the remaining members of the TLR family from human and mouse, including TLR7, 8, and 9.     

The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.     

The phosphotyrosine peptide binding specificity of Nck1 and Nck2 Src homology 2 domains

Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nckalpha) and Nck2 (Nckbeta and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1.     

The application of modular protein domains in proteomics

The ability of modular protein domains to independently fold and bind short peptide ligands both in vivo and in vitro has allowed a significant number of protein-protein interaction studies to take advantage of them as affinity and detection reagents. Here, we refer to modular domain based proteomics as "domainomics" to draw attention to the potential of using domains and their motifs as tools in proteomics. In this review we describe core concepts of domainomics, established and emerging technologies, and recent studies by functional category. Accumulation of domain-motif binding data should ultimately provide the foundation for domain-specific interactomes, which will likely reveal the underlying substructure of protein networks as well as the selectivity and plasticity of signal transduction.     

Functional genomics of intracellular peptide recognition domains with combinatorial biology methods

Phage-displayed peptide libraries have been used to identify specific ligands for peptide-binding domains that mediate intracellular protein-protein interactions. These studies have provided significant insights into the specificities of particular domains. For PDZ domains that recognize C-terminal sequences, the information has proven useful in identifying natural binding partners from genomic databases. For SH3 domains that recognize internal proline-rich motifs, the results of database searches with phage-derived ligands have been compared with the results of yeast-two-hybrid experiments to produce overlap networks that reliably predict natural protein-protein interactions. In addition, libraries of phage-displayed PDZ and SH3 domains have been used to identify the residues responsible for ligand recognition, and also to engineer domains with altered specificities.     

Implementation of communication‐mediating domains for non‐ribosomal peptide production in Saccharomyces cerevisiae

Saccharomyces cerevisiae has in several cases been proven to be a suitable host for the production of natural products and was recently exploited for the production of non‐ribosomal peptides. Synthesis of non‐ribosomal peptides (NRPs) is mediated by NRP synthetases (NRPSs), modular enzymes, which are often organized in enzyme complexes. In these complexes, partner NRPSs interact via communication‐mediating domains (COM domains). In order to test whether functional interaction between separate NRPS modules is possible in yeast we constructed a yeast strain expressing two modules with compatible COM domains from two plasmids. Successful production as well as secretion of the expected dipeptide was detected. This opens the possibility of using yeast as a eukaryotic platform for fast assessment of new module combinations for the development of novel NRP compounds. Biotechnol. Bioeng. 2010;106: 841–844. © 2010 Wiley Periodicals, Inc.     

The modular structure of ThDP‐dependent enzymes

Thiamine diphosphate (ThDP)‐dependent enzymes form a diverse protein family which was classified into nine superfamilies. The cofactor ThDP is bound at the interface between two catalytic domains, the PYR and the PP domain. The nine superfamilies were assigned to five different structural architectures. Two superfamilies, the sulfopyruvate decarboxylases and α‐ketoacid dehydrogenases 2, consist of separate PYR and PP domains. The oxidoreductase superfamily is of the intra‐monomer/PYR‐PP type with an N‐terminal PYR and a subsequent PP domain. The active enzymes form homodimers with the ThDP cofactor bound at the interface between a PYR and a PP domain of the same monomer. Decarboxylases are of the inter‐monomer/PYR‐PP type with the cofactor bound between domains from different monomers. 1‐Deoxy‐d ‐xylulose‐5‐phosphate synthases are of the intra‐monomer/PP‐PYR type. The transketolases, α‐ketoglutarate dehydrogenases, and α‐ketoacid dehydrogenases 1 are of the inter‐monomer/PP‐PYR type. For the phosphonopyruvate decarboxylases, definitive assessment of the structural architecture is not possible due to lack of structure information. By applying a structure‐based domain alignment method, sequences of more than 62,000 PYR and PP domains were identified and aligned. Although the sequence similarity of the catalytic domains is low between different superfamilies, seven positions were identified to be highly conserved, including the cofactor binding GDGX_24,27N motif, the cofactor‐activating glutamic acid, and two structurally equivalent glycines in both the PYR and the PP domain. An evolutionary pathway of ThDP‐dependent enzymes is proposed which explains the sequence and structure diversity of this family by three basic evolutionary events: domain recruitment, domain linkage, and structural rearrangement of catalytic domains. Proteins 2014; 82:2523–2537. © 2014 Wiley Periodicals, Inc.     

Solution structure of PCP, a prototype for the peptidyl carrier domains of modular peptide synthetases

BACKGROUND: Nonribosomal peptide synthetases (NRPSs) are large modular enzymes responsible for the synthesis of a variety of microbial bioactive peptides. They consist of modules that each recognise and incorporate one specific amino acid into the peptide product. A module comprises several domains, which carry out the individual reaction steps. After activation by the adenylation domain, the amino acid substrate is covalently tethered to a 4'-phosphopantetheinyl cofactor of a peptidyl carrier domain (PCP) that passes the substrate to the reaction centres of the consecutive domains. RESULTS: The solution structure of PCP, a distinct peptidyl carrier protein derived from the equivalent domain of an NRPS, was solved using NMR techniques. PCP is a distorted four-helix bundle with an extended loop between the first two helices. Its overall fold resembles the topology of acyl carrier proteins (ACPs) from Escherichia coli fatty acid synthase and actinorhodin polyketide synthase from Streptomyces coelicolor; however, the surface polarity and the length and relative alignment of the helices are different. The conserved serine, which is the cofactor-binding site, has the same location as in the ACPs and is situated within a stretch of seven flexible residues. CONCLUSIONS: The structure of PCP reflects its character as a protein domain. The fold is well defined between residues 8 and 82 and the structural core of the PCP domain can now be defined as a region spanning 37 amino acids in both directions from the conserved serine. The flexibility of the post-translationally modified site might have implications for interactions with the cooperating proteins or NRPS domains.     

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.     

Structural insights into specificity and diversity in mechanisms of ubiquitin recognition by ubiquitin-binding domains

UBDs [Ub (ubiquitin)-binding domains], which are typically small protein motifs of <50 residues, are used by receptor proteins to transduce post-translational Ub modifications in a wide range of biological processes, including NF-κB (nuclear factor κB) signalling and proteasomal degradation pathways. More than 20 families of UBDs have now been characterized in structural detail and, although many recognize the canonical Ile44/Val70-binding patch on Ub, a smaller number have alternative Ub-recognition sites. The A20 Znf (A20-like zinc finger) of the ZNF216 protein is one of the latter and binds with high affinity to a polar site on Ub centred around Asp58/Gln62. ZNF216 shares some biological function with p62, with both linked to NF-κB signal activation and as shuttle proteins in proteasomal degradation pathways. The UBA domain (Ub-associated domain) of p62, although binding to Ub through the Ile44/Val70 patch, is unique in forming a stable dimer that negatively regulates Ub recognition. We show that the A20 Znf and UBA domain are able to form a ternary complex through independent interactions with a single Ub molecule, supporting functional models for Ub as a 'hub' for mediating multi-protein complex assembly and for enhancing signalling specificity.