Amino Acid Residues For Binding Research Articles

Structural characterization of Fis — A transcriptional regulator from pathogenic Pasteurella multocida essential for expression of virulence factors

Pasteurella multocida is responsible behind a variety of diseases in animals. The disease causing substance of this bacterium is a capsular polysaccharide. The expression of the gene that codes for the bacterial capsule is regulated by the protein Fis. Fis also regulates the expression of various different genes in P. multocida. So far there have been no previous reports that depict the characterization of Fis from P. multocida from a structural point of view. In the present work, an attempt has been made to characterize Fis by in silico methods. The structure of Fis was built by comparative modeling technique. The model of Fis was then docked onto the corresponding promoter regions of the gene encoding the capsular polysaccharide. The docked complexes of promoter DNA with Fis protein were subjected to molecular dynamics simulations to identify the mode of DNA–protein interactions. The DNA binding amino acid residues from the Fis protein were identified. And a mechanistic detail of the DNA binding interactions was predicted. So far, this is the first report that depicts the mechanistic details of Fis–DNA interactions involved in the regulation of gene expression by Fis protein. This work may therefore be useful to illuminate the still obscure molecular mechanism behind the disease propagation by P. multocida.

Carbohydrate-binding protein identification by coupling structural similarity searching with binding affinity prediction.

Carbohydrate-binding proteins (CBPs) are potential biomarkers and drug targets. However, the interactions between carbohydrates and proteins are challenging to study experimentally and computationally because of their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates as exists in RNA, DNA, and proteins. Here, we describe a structure-based function-prediction technique called SPOT-Struc that identifies carbohydrate-recognizing proteins and their binding amino acid residues by structural alignment program SPalign and binding affinity scoring according to a knowledge-based statistical potential based on the distance-scaled finite-ideal gas reference state (DFIRE). The leave-one-out cross-validation of the method on 113 carbohydrate-binding domains and 3442 noncarbohydrate binding proteins yields a Matthews correlation coefficient of 0.56 for SPalign alone and 0.63 for SPOT-Struc (SPalign + binding affinity scoring) for CBP prediction. SPOT-Struc is a technique with high positive predictive value (79% correct predictions in all positive CBP predictions) with a reasonable sensitivity (52% positive predictions in all CBPs). The sensitivity of the method was changed slightly when applied to 31 APO (unbound) structures found in the protein databank (14/31 for APO versus 15/31 for HOLO). The result of SPOT-Struc will not change significantly if highly homologous templates were used. SPOT-Struc predicted 19 out of 2076 structural genome targets as CBPs. In particular, one uncharacterized protein in Bacillus subtilis (1oq1A) was matched to galectin-9 from Mus musculus. Thus, SPOT-Struc is useful for uncovering novel carbohydrate-binding proteins. SPOT-Struc is available at http://sparks-lab.org.

Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus

Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway.

Modelling, substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA.

The AzgA purine/H(+) symporter of Aspergillus nidulans is the founding member of a functionally and phylogenetically distinct transporter family present in fungi, bacteria and plants. Here a valid AzgA topological model is built based on the crystal structure of the Escherichia coli uracil transporter UraA, a member of the nucleobase-ascorbate transporter (NAT/NCS2) family. The model consists of 14 transmembrane, mostly α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct compact core of 8 TMSs, made of two intertwined inverted repeats (TMSs 1-4 and 8-11), is topologically distinct from a flexible domain (TMSs 5-7 and 12-14). A putative substrate binding cavity is visible between the core and the gate domains. Substrate docking, molecular dynamics and mutational analysis identified several residues critical for purine binding and/or transport in TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339 (TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates, while Asp342 (TMS8) might be involved in subsequent substrate translocation, through H(+) binding and symport. Thus, AzgA and other NAT transporters use topologically similar TMSs and amino acid residues for substrate binding and transport, which in turn implies that AzgA-like proteins constitute a distant subgroup of the ubiquitous NAT family.

Prediction of binding affinities of PCDDs, PCDFs and PCBs using docking-based Comparative Molecular Similarity Indices Analysis

Polychlorinated Dibenzodioxins (PCDDs), Dibenzofurans (PCDFs) and Biphenyls (PCBs) are industrial compounds or byproducts that can cause toxic effects after binding to aryl hydrocarbon receptor (AhR). But the mechanism about PCDDs, PCDFs and PCBs binding to AhR is unclear. To study the interaction and significant amino acid residues in binding of PCDDs, PCDFs and PCBs to AhR, a docking-based Comparative Molecular Similarity Indices Analysis (CoMSIA) was performed on a set of structurally diverse PCDDs, PCDFs and PCBs with known binding affinities. The docking-based CoMSIA model (non-cross-validated regression coefficient of 0.942 and cross-validated regression coefficient of 0.768) was developed and compared with previous report, the presented docking-based CoMSIA model showed good robustness and predictive performance. The obtained docking conformations and predictive CoMSIA model could provide clues to understand key residues and interactions between receptor and compounds of interest.

Predicting the function of 4-coumarate:CoA ligase (LJ4CL1) in Lonicera japonica.

4-Coumarate:CoA ligases (4CLs) are a group of essential enzymes involved in the pathway of phenylpropanoid-derived compound metabolisms; however it is still difficult to identify orthologs and paralogs of these important enzymes just based on sequence similarity of the conserved domains. Using sequence data of 20 plant species from the public databases and sequences from Lonicera japonica, we define 1252 adenosine monophosphate (AMP)-dependent synthetase/ligase sequences and classify them into three phylogenetic clades. 4CLs are in one of the four subgroups, according to their partitioning, with known proteins characterized in A. thaliana and Oryza sativa. We also defined 184 non-redundant sequences that encode proteins containing the GEICIRG motif and the taxonomic distribution of these GEICIRG-containing proteins suggests unique catalytic activities in plants. We further analyzed their transcription levels in L. japonica and L. japonica. var. chinensis flowers and chose the highest expressed genes representing the subgroups for structure and binding site predictions. Coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of the L. japonica flowers, the structural study on putative substrate binding amino acid residues, ferulate, and 4-coumaric acid of the conserved binding-site of LJ4CL1 leads to a conclusion that this highly expressed protein group in the flowers may process 4-coumarate that represents 90% of the known phenylpropanoid-derived compounds. The activity of purified crude LJ4CL1 protein was analyzed using 4-coumarate as template and high activity indicating that 4-coumarate is one of the substrates of LJ4CL1.

In-silico analysis of heat shock protein 47 for identifying the novel therapeutic agents in the management of oral submucous fibrosis.

Heat shock proteins-47 (HSP47) is a collagen specific molecular chaperone, involved in the processing and/or secretion of procollagen. It seems to be regularly upregulated in various fibrotic or collagen disorders. Hence, this protein can be a potential target for the treatment of various fibrotic diseases including oral submucous fibrosis (OSF), which is a collagen metabolic disorder of oral cavity and whose etiopathogeneic mechanism and therapeutic protocols are still not well documented. The aim of this study is to identify the novel therapeutic agents using in-silico methods for the management of OSF. The objectives of this study are to identify the binding sites of HSP47 on the collagen molecule and to identify the lead compound with anti-HSP47 activity from the library of natural compounds, using in-silico methodology. The web-based and tool based in-silico analysis of the HSP47 and collagen molecules are used in this study. The crystal structure of collagen and HSP47 were retrieved from Protein Data Bank website. The binding site identification and the docking studies are done using Molegro Virtual Docker offline tool. Out of the 104 Natural compounds, six ligands are found to possess best binding affinity to the binding amino acid residues. Silymarin binds with the 4AU2A receptor and the energy value are found to be -178.193 with four Hbonds. The other best five natural compounds are hesperidin, ginkgolides, withanolides, resveratrol, and gingerol. Our findings provide the basis for the in-vitro validation of the above specified compounds, which can possibly act as "lead" molecules in designing the drugs for OSF. HSP47 can be a potential candidate to target, in order to control the production of abundance collagen in OSF. Hence, the binding sites of HSP47 with collagen are identified and some natural compounds with a potential to bind with these binding receptors are also recognized. These natural compounds might act as anti-HSP47 lead molecules in designing novel therapeutic agents for OSF, which are so far unavailable.

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Based on sequence alignment of selected Cl− dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and BlTreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified BlTreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of BlTreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of BlTreA.

Rainbow trout peptidoglycan recognition protein has an anti-inflammatory function in liver cells

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are structurally conserved through evolution in both invertebrate and vertebrate animals. PGRPs exert diverse host-defense functions both through direct antibacterial activity and through indirect effects, including the induction of antimicrobial peptides and the modulation of inflammation and immune responses. In this study, we identified the gene encoding a long form of PGRP (OmPGRP-L1) from the rainbow trout, Oncorhynchus mykiss, and investigated whether it has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 challenged with fish pathogenic bacteria. OmPGRP-L1 contains the conserved PGRP domain and the four Zn2+-binding amino acid residues required for amidase activity. In RTH-149 cells, OmPGRP-L1 expression was increased by bacterial stimulation. Loss-of-function and gain-of-function experiments indicated that OmPGRP-L1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmPGRP-L1 in RTH-149 cells challenged with Edwardsiella tarda dramatically increased the expression of IL-1β and TNF-α. In contrast, overexpression of OmPGRP-L1 or its amidase-inactive mutant OmPGRP-L1(C472S) resulted in down-regulation of IL-1β and TNF-α expression. When overexpressed in RTH-149 cells, OmPGRP-L1 inhibited NF-κB activity with or without bacterial stimulation. Collectively, these findings suggest that OmPGRP-L1 has an anti-inflammatory function, independent of its amidase activity, possibly via NF-κB inhibition in liver cells.

Engineering Plant Disease Resistance Based on TAL Effectors

Transcription activator-like (TAL) effectors are encoded by plant-pathogenic bacteria and induce expression of plant host genes. TAL effectors bind DNA on the basis of a unique code that specifies binding of amino acid residues in repeat units to particular DNA bases in a one-to-one correspondence. This code can be used to predict binding sites of natural TAL effectors and to design novel synthetic DNA-binding domains for targeted genome manipulation. Natural mechanisms of resistance in plants against TAL effector-containing pathogens have given insights into new strategies for disease control.

Strategic targeting of multiple water-mediated interactions: a concise and rational structure-based design approach to potent and selective MMP-13 inhibitors.

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

The interaction of Kinesin-1 with its adaptor protein JIP1 can be regulated via proteins binding to the JIP1-PTB domain

BackgroundThe regulatory mechanisms of motor protein-dependent intracellular transport are still not fully understood. The kinesin-1-binding protein, JIP1, can function as an adaptor protein that links kinesin-1 and other JIP1-binding “cargo” proteins. However, it is unknown whether these “cargo” proteins influence the JIP1–kinesin-1 binding.ResultsWe show here that JIP1–kinesin-1 binding in Neuro2a cells was dependent on conserved amino acid residues in the JIP1-phosphotyrosine binding (PTB) domain, including F687. In addition, mutation of F687 severely affected the neurite tip localization of JIP1. Proteomic analysis revealed another kinesin-1 binding protein, JIP3, as a major JIP1 binding protein. The association between JIP1 and JIP3 was dependent on the F687 residue in JIP1, and this association induced the formation of a stable ternary complex with kinesin-1. On the other hand, the binding of JIP1 and JIP3 was independent of kinesin-1 binding. We also show that other PTB binding proteins can interrupt the formation of the ternary complex.ConclusionsThe formation of the JIP1–kinesin-1 complex depends on the protein binding-status of the JIP1 PTB domain. This may imply a regulatory mechanism of kinesin-1-dependent intracellular transport.

The transcriptional repressor activity of ASYMMETRIC LEAVES1 is inhibited by direct interaction with calmodulin in Arabidopsis

Calmodulin (CaM), a key Ca2+ sensor, regulates diverse cellular processes by modulating the activity of a variety of enzymes and proteins. However, little is known about the biological function of CaM in plant development. In this study, an ASYMMETRIC LEAVES1 (AS1) transcription factor was isolated as a CaM-binding protein. AS1 contains two putative CaM-binding domains (CaMBDs) at the N-terminus. Using domain mapping analysis, both predicted domains were identified as authentic Ca2+ -dependent CaMBDs. We identified three hydrophobic amino acid residues for CaM binding, Trp49 in CaMBDI, and Trp81 and Phe103 in CaMBDII. The interactions of AS1 with CaM were verified in yeast and plant cells. Based on electrophoretic mobility shift assays, CaM inhibited the DNA-binding activity of the AS1/AS2 complex to two cis-regulatory motifs in the KNAT1 promoter. Furthermore, CaM relieved the suppression of KNAT1 transcription by AS1 not only in transient expression assays of protoplasts but also by the overexpression of a CaM-binding negative form of AS1 in as1 mutant plant. Our study suggests that CaM, a calcium sensor, can be involved in the transcriptional control of meristem cell-specific genes by the inhibition of AS1 under the condition of higher levels of Ca2+ in plants.

Residue Y161 of Influenza Virus Hemagglutinin Is Involved in Viral Recognition of Sialylated Complexes from Different Hosts

Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to N-acetylneuraminic acid α3 (NeuAcα3) sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species-specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied, and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved HA surface-located amino acid residues in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell-type-dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharide binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to N-glycolylneuraminic acid (NeuGc). Rescued mutant Y161A viruses demonstrated a 5- to 10-fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism.

A cross-protective mAb recognizes a novel epitope within the flavivirus NS1 protein

Despite a resurgence of flavivirus infections worldwide, no approved therapeutic agent exists for any member of the genus. While cross-reactive antibodies with therapeutic potential against flaviviruses have been generated, the majority of them are anti-E antibodies with the potential to cause antibody-dependent enhancement of flavivirus infection and disease. We described previously mAbs against the non-structural NS1 protein of the West Nile virus (WNV) that were protective in mice when administered pre- or post-infection of WNV. Here, we demonstrate that one of these mAbs (16NS1) cross-reacted with Japanese encephalitis virus (JEV) and exhibited protective activity against a lethal JEV infection. Overlapping peptide mapping analysis combined with site-specific mutations identified a novel epitope ¹¹⁶KAWGKSILFA¹²⁵ and critical amino acid residues (¹¹⁸W and ¹²²I) for 16NS1 mAb binding. These results may facilitate the development of a broadly therapeutic mAb that lacks enhancing potential and/or subunit-based vaccine against flaviviruses that target the NS1 protein.

Use of TILLING and robotised enzyme assays to generate an allelic series of Arabidopsis thaliana mutants with altered ADP-glucose pyrophosphorylase activity

ADP-glucose pyrophosphorylase (AGPase) catalyses the synthesis of ADP-glucose, and is a highly regulated enzyme in the pathway of starch synthesis. In Arabidopsis thaliana, the enzyme is a heterotetramer, containing two small subunits encoded by the APS1 gene and two large subunits encoded by the APL1-4 genes. TILLING ( Targeting Induced Local Lesions IN Genomes) of a chemically mutagenised population of A. thaliana plants identified 33 novel mutations in the APS1 gene, including 21 missense mutations in the protein coding region. High throughput measurements using a robotised cycling assay showed that maximal AGPase activity in the aps1 mutants varied from <15 to 117% of wild type (WT), and that the kinetic properties of the enzyme were altered in several lines, indicating a role for the substituted amino acid residues in catalysis or substrate binding. These results validate the concept of using such a platform for efficient high-throughput screening of very large populations of mutants, natural accessions or introgression lines. AGPase was estimated to have a flux control coefficient of 0.20, indicating that the enzyme exerted only modest control over the rate of starch synthesis in plants grown under short day conditions (8 h light/16 h dark) with an irradiance of 150 μmol quanta m −2 s −1. Redox activation of the enzyme, via reduction of the intermolecular disulphide bridge between the two small subunits, was increased in several lines. This was sometimes, but not always, associated with a decrease in the abundance of the APS1 protein. In conclusion, the TILLING technique was used to generate an allelic series of aps1 mutants in A. thaliana that revealed new insights into the multi-layered regulation of AGPase. These mutants offer some advantages over the available loss-of-function mutants, e.g. adg1, for investigating the effects of subtle changes in the enzyme's activity on the rate of starch synthesis.

Heterologous expression of two FAD-dependent oxidases with (S)-tetrahydroprotoberberine oxidase activity from Arge mone mexicana and Berberis wilsoniae in insect cells

Berberine, palmatine and dehydrocoreximine are end products of protoberberine biosynthesis. These quaternary protoberberines are elicitor inducible and, like other phytoalexins, are highly oxidized. The oxidative potential of these compounds is derived from a diverse array of biosynthetic steps involving hydroxylation, intra-molecular C-C coupling, methylenedioxy bridge formation and a dehydrogenation reaction as the final step in the biosynthesis. For the berberine biosynthetic pathway, the identification of the dehydrogenase gene is the last remaining uncharacterized step in the elucidation of the biosynthesis at the gene level. An enzyme able to catalyze these reactions, (S)-tetrahydroprotoberberine oxidase (STOX, EC 1.3.3.8), was originally purified in the 1980s from suspension cells of Berberis wilsoniae and identified as a flavoprotein (Amann et al. 1984). We report enzymatic activity from recombinant STOX expressed in Spodoptera frugiperda Sf9 insect cells. The coding sequence was derived successively from peptide sequences of purified STOX protein. Furthermore, a recombinant oxidase with protoberberine dehydrogenase activity was obtained from a cDNA library of Argemone mexicana, a traditional medicinal plant that contains protoberberine alkaloids. The relationship of the two enzymes is discussed regarding their enzymatic activity, phylogeny and the alkaloid occurrence in the plants. Potential substrate binding and STOX-specific amino acid residues were identified based on sequence analysis and homology modeling.

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity

Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains.