Structural basis for binding of porphyrin to human telomeres

Maintenance of telomere integrity is a hallmark of human cancer, and the single-stranded 3' ends of telomeric DNA are targets for small-molecule anticancer therapies. We report here the crystal structure of a bimolecular human telomeric quadruplex, of the sequence d(TAGGGTTAGGG), in a complex with the quadruplex-binding ligand 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) to a resolution of 2.09 A. The DNA quadruplex topology is parallel-stranded with external double-chain-reversal propeller loops, consistent with previous structural determinations. The porphyrin molecules bind by stacking onto the TTA nucleotides, either as part of the external loop structure or at the 5' region of the stacked quadruplex. This involves stacked on hydrogen-bonded base pairs, formed from those nucleotides not involved in the formation of G-tetrads, and there are thus no direct ligand interactions with G-tetrads. This is in accord with the relative nonselectivity by TMPyP4 for quadruplex DNAs compared to duplex DNA. Porphyrin binding is achieved by remodeling of loops compared to the ligand-free structures. Implications for the design of quadruplex-binding ligands are discussed, together with a model for the formation of anaphase bridges, which are observed following cellular treatment with TMPyP4.     

Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Structural basis for high-affinity binding of LEDGF PWWP to mononucleosomes

Lens epithelium-derived growth factor (LEDGF/p75) tethers lentiviral preintegration complexes (PICs) to chromatin and is essential for effective HIV-1 replication. LEDGF/p75 interactions with lentiviral integrases are well characterized, but the structural basis for how LEDGF/p75 engages chromatin is unknown. We demonstrate that cellular LEDGF/p75 is tightly bound to mononucleosomes (MNs). Our proteomic experiments indicate that this interaction is direct and not mediated by other cellular factors. We determined the solution structure of LEDGF PWWP and monitored binding to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA by NMR. Results reveal two distinct functional interfaces of LEDGF PWWP: a well-defined hydrophobic cavity, which selectively interacts with the H3K36me3 peptide and adjacent basic surface, which non-specifically binds DNA. LEDGF PWWP exhibits nanomolar binding affinity to purified native MNs, but displays markedly lower affinities for the isolated H3K36me3 peptide and DNA. Furthermore, we show that LEDGF PWWP preferentially and tightly binds to in vitro reconstituted MNs containing a tri-methyl-lysine analogue at position 36 of H3 and not to their unmodified counterparts. We conclude that cooperative binding of the hydrophobic cavity and basic surface to the cognate histone peptide and DNA wrapped in MNs is essential for high-affinity binding to chromatin.     

Structural and functional basis for ADP-ribose and poly(ADP-ribose) binding by viral macro domains

Macro domains constitute a protein module family found associated with specific histones and proteins involved in chromatin metabolism. In addition, a small number of animal RNA viruses, such as corona- and toroviruses, alphaviruses, and hepatitis E virus, encode macro domains for which, however, structural and functional information is extremely limited. Here, we characterized the macro domains from hepatitis E virus, Semliki Forest virus, and severe acute respiratory syndrome coronavirus (SARS-CoV). The crystal structure of the SARS-CoV macro domain was determined at 1.8-Angstroms resolution in complex with ADP-ribose. Information derived from structural, mutational, and sequence analyses suggests a close phylogenetic and, most probably, functional relationship between viral and cellular macro domain homologs. The data revealed that viral macro domains have relatively poor ADP-ribose 1"-phosphohydrolase activities (which were previously proposed to be their biologically relevant function) but bind efficiently free and poly(ADP-ribose) polymerase 1-bound poly(ADP-ribose) in vitro. Collectively, these results suggest to further evaluate the role of viral macro domains in host response to viral infection.     

Structural basis for NHERF1 PDZ domain binding

The Na(+)/H(+) exchange regulatory factor-1 (NHERF1) is a scaffolding protein that possesses two tandem PDZ domains and a carboxy-terminal ezrin-binding domain (EBD). The parathyroid hormone receptor (PTHR), type II sodium-dependent phosphate cotransporter (Npt2a), and β2-adrenergic receptor (β2-AR), through their respective carboxy-terminal PDZ-recognition motifs, individually interact with NHERF1 forming a complex with one of the PDZ domains. In the basal state, NHERF1 adopts a self-inhibited conformation, in which its carboxy-terminal PDZ ligand interacts with PDZ2. We applied molecular dynamics (MD) simulations to uncover the structural and biochemical basis for the binding selectivity of NHERF1 PDZ domains. PDZ1 uniquely forms several contacts not present in PDZ2 that further stabilize PDZ1 interactions with target ligands. The binding free energy (ΔG) of PDZ1 and PDZ2 with the carboxy-terminal, five-amino acid residues that form the PDZ-recognition motif of PTHR, Npt2a, and β2-AR was calculated and compared with the calculated ΔG for the self-association of NHERF1. The results suggest that the interaction of the PTHR, β2-adrenergic, and Npt2a involves competition between NHERF1 PDZ domains and the target proteins. The binding of PDZ2 with PTHR may also compete with the self-inhibited conformation of NHERF1, thereby contributing to the stabilization of an active NHERF1 conformation.     

Structural basis of human cytoglobin for ligand binding

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.     

Structural basis for induced-fit binding of Rho-kinase to the inhibitor Y-27632

Rho-kinase is a main player in the regulation of cytoskeletal events and a promising drug target in the treatment of both vascular and neurological disorders. Here we report the crystal structure of the Rho-kinase catalytic domain in complex with the specific inhibitor Y-27632. Comparison with the structure of PKA bound to this inhibitor revealed a potential induced-fit binding mode that can be accommodated by the phosphate binding loop. This binding mode resembles to that observed in the Rho-kinase-fasudil complex. A structural database search indicated that a pocket underneath the phosphate-binding loop is present that favors binding to a small aromatic ring. Introduction of such a ring group might spawn a new modification scheme of pre-existing protein kinase inhibitors for improved binding capability.     

Structural basis for selective GABA binding in bacterial pathogens

GABA acts as an intercellular signal in eukaryotes and as an interspecies signal in host–microbe interactions. Structural characteristics of selective eukaryotic GABA receptors and bacterial GABA sensors are unknown. Here, we identified the selective GABA‐binding protein, called Atu4243, in the plant pathogen Agrobacterium tumefaciens. A constructed atu4243 mutant was affected in GABA transport and in expression of the GABA‐regulated functions, including aggressiveness on two plant hosts and degradation of the quorum‐sensing signal. The GABA‐bound Atu4243 structure at 1.28 Å reveals that GABA adopts a conformation never observed so far and interacts with two key residues, Arg²⁰³ and Asp²²⁶ of which the role in GABA binding and GABA signalling in Agrobacterium has been validated using appropriate mutants. The conformational GABA‐analogue trans‐4‐aminocrotonic acid (TACA) antagonizes GABA activity, suggesting structural similarities between the binding sites of the bacterial sensor Atu4243 and mammalian GABA_C receptors. Exploration of genomic databases reveals Atu4243 orthologues in several pathogenic and symbiotic proteobacteria, such as Rhizobium, Azospirillum, Burkholderia and Pseudomonas. Thus, this study establishes a structural basis for selective GABA sensors and offers opportunities for deciphering the role of the GABA‐mediated communication in several host–pathogen interactions.     

Structural basis of heparin binding to camel peptidoglycan recognition protein-S

Short peptidoglycan recognition protein (PGRP-S) is a member of the innate immunity system in mammals. PGRP-S from Camelus dromedarius (CPGRP-S) is found to be highly potent against bacterial infections. It is capable of binding to a wide range of pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). The heparin-like polysaccharides have also been observed in some bacteria such as the capsule of K5 Escherichia coli thus making them relevant for determining the nature of their interactions with CPGRP-S. The binding studies of CPGRP-S with heparin disaccharide in solution using surface plasmon resonance gave a value 3.3×10^-7 M for the dissociation constant (Kd). The structure of the heparin bound CPGRP-S determined at 2.8Å resolution revealed the presence of a bound heparin molecule in the binding pocket of CPGRP-S. It was found anchored tightly to the protein with the help of several ionic and hydrogen bonded interactions. Three sulphate groups of heparin S1, S2 and S3 have been found to interact with residues, Arg-31, Lys-90, Thr- 97, Asn-99 Asn-140, Gln-150 and Arg-170 of CPGRP-S. The binding site includes two subsites, S-I and S-II with cleft-like structures. Heparin disaccharide is bound in subsite S-I. Previously determined structures of the complexes of CPGRP-S with LPS, LTA and PGN also showed that their glycan moieties were also held in subsite S-I indicating that heparin disaccharide also represents an important element for the recognition by CPGRP-S.     

Structural basis for triclosan and NAD binding to enoyl-ACP reductase of Plasmodium falciparum

Recent discovery of type II fatty acid synthase in the malarial parasite Plasmodium falciparum responsible for the most debilitating form of the disease in humans makes it ideal as a target for the development of novel antimalarials. Also, the identification of the enoyl-acyl carrier protein reductase from P. falciparum and the demonstration of its inhibition by triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol], a potent antibacterial compound, provide strong support for the above. In the studies reported here, a model of the enzyme in complex with triclosan and the cofactor NAD has been built by homology modeling with a view to understand its binding properties and to explore the potential of triclosan as a lead compound in designing effective antimalarial drugs. The model indeed provided the structural rationale for its interaction with ligands and the cofactor and revealed unique characteristics of its binding site which could be exploited for improving the specificity of the inhibitors.     

Structural basis for distinct binding properties of the human galectins to Thomsen-Friedenreich antigen

The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif ((51)AHGDA(55)) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins.     

Structural basis for antagonism by suramin of heparin binding to vaccinia complement protein

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.     

Structural basis for binding of cyclic 2-oxoglutarate analogues to factor-inhibiting hypoxia-inducible factor

Aromatic analogues of the 2-oxoglutarate co-substrate of the hypoxia-inducible factor hydroxylases are shown to bind at the active site iron: Pyridine-2,4-dicarboxylate binds as anticipated with a single molecule chelating the iron in a bidentate manner. The binding mode of a hydroxamic acid analogue, at least in the crystalline state, is unusual because two molecules of the inhibitor are observed at the active site and partial displacement of the iron binding aspartyl residue was observed.     

Structural basis for the receptor binding specificity of Norwalk virus

Noroviruses are positive-sense, single-stranded RNA viruses that cause acute gastroenteritis. They recognize human histo-blood group antigens as receptors in a strain-specific manner. The structures presented here were analyzed in order to elucidate the structural basis for differences in ligand recognition of noroviruses from different genogroups, the prototypic Norwalk virus (NV; GI-1) and VA387 (GII-4), which recognize the same A antigen but differ in that NV is unable to bind to the B antigen. Two forms of the receptor-binding domain of the norovirus coat protein, the P domain and the P polypeptide, that were previously shown to differ in receptor binding and P-particle formation properties were studied. Comparison of the structures of the NV P domain with and without A trisaccharide and the NV P polypeptide revealed no major ligand-induced changes. The 2.3-A cocrystal structure reveals that the A trisaccharide binds to the NV P domain through interactions with the residues Ser377, Asp327, His329, and Ser380 in a mode distinct from that previously reported for the VA387 P-domain-A-trisaccharide complex. Mutational analyses confirm the importance of these residues in NV P-particle binding to native A antigen. The alpha-GalNAc residue unique to the A trisaccharide is buried deeply in the NV binding pocket, unlike in the structures of A and B trisaccharides bound to VA387 P domain, where the alpha-fucose residue forms the most protein contacts. The A-trisaccharide binding mode seen in the NV P domain complex cannot be sterically accommodated in the VA387 P domain.     

Structural basis for tetrodotoxin-resistant sodium channel binding by mu-conotoxin SmIIIA

SmIIIA is a new micro-conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other micro-conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has several distinctive features, including the absence of hydroxyproline, and is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be characterized. It therefore represents a potentially useful tool to investigate the functional roles of these channels. We have determined the three-dimensional structure of SmIIIA in aqueous solution. Consistent with the absence of hydroxyprolines, SmIIIA adopts a single conformation with all peptide bonds in the trans configuration. The spatial orientations of several conserved Arg and Lys side chains, including Arg14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other micro-conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in micro-conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other micro-conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp15 side chain, which is unique to SmIIIA. Arg17, which replaces Lys in the other micro-conotoxins, may also be important. Consistent with these inferences from the structure, assays of two chimeras of SmIIIA and PIIIA in which their N- and C-terminal halves were recombined, indicated that residues in the C-terminal half of SmIIIA confer affinity for tetrodotoxin-resistant sodium channels in the cell bodies of frog sympathetic neurons. SmIIIA and the chimera possessing the C-terminal half of SmIIIA also inhibit tetrodotoxin-resistant sodium channels in the postganglionic axons of sympathetic neurons, as indicated by their inhibition of C-neuron compound action potentials that persist in the presence of tetrodotoxin.     

Structural basis for HLA-DQ binding by the streptococcal superantigen SSA

Streptococcal superantigen (SSA) is a 28,000 Mr toxin originally isolated from a pathogenic strain of Streptococcus pyogenes that has 60% sequence identity with staphylococcal enterotoxin B (SEB). SSA and SEB, however, do not compete for binding on the surfaces of cells expressing MHC class II molecules. This behavior had been ascribed to SSA and SEB binding to distinct sites on, or different subsets of, HLA-DR molecules. Here we demonstrate that SSA binds predominantly to HLA-DQ, rather than to HLA-DR molecules, and present the crystal structure of SSA at 1.85 A resolution. These data provide a structural basis for interpreting the interaction of SSA with HLA-DQ molecules as well as a foundation for understanding bacterial superantigen affinities for distinct MHC isotypes.