Large Hydrophobic Pocket Research Articles

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.

Crystal Structure and Biochemical Features of EfeB/YcdB from Escherichia coli O157: ASP235 PLAYS DIVERGENT ROLES IN DIFFERENT ENZYME-CATALYZED PROCESSES

EfeB/YcdB is a member of the dye-decolorizing peroxidase (DyP) protein family. A recent study has shown that this protein can extract iron from heme without breaking the tetrapyrrole ring. We report the crystal structure of EfeB from Escherichia coli O157 bound to heme at 1.95 Å resolution. The EfeB monomer contains two domains. The heme molecule is located in a large hydrophobic pocket in the C-terminal domain. A long loop connecting the two domains extensively interacts with the heme, which is a distinctive structural feature of EfeB homologues. A large tunnel formed by this loop and the β-sheet of C-terminal domain provides a potential cofactor/substrate binding site. Biochemical data show that the production of protoporphyrin IX (PPIX) is closely related to the peroxidation activity. The mutant D235N keeps nearly the same activity of guaiacol peroxidase as the wild-type protein, whereas the corresponding mutation in the classic DyP protein family completely abolished the peroxidation activity. These results suggest that EfeB is a unique member of the DyP protein family. In addition, dramatically enhanced fluorescence excitation and emission of EfeB-PPIX was observed, implying this protein may be used as a red color fluorescence marker.

The structural flexibility of the shank1 PDZ domain is important for its binding to different ligands

The PDZ domain of the shank protein interacts with numerous cell membrane receptors and cytosolic proteins via the loosely defined binding motif X-(Ser/Thr)-X-Φ-COOH (Φ represents hydrophobic residues) at the carboxyl terminus of its target protein. This enables shank to serve as a membrane-associated scaffold for the assembly of signaling complexes. As the list of proteins that bind to the shank PDZ domain grows, it is not immediately clear what structural element(s) mediate this domain’s target specificity or the plasticity required to bind its different targets. Here, we have determined the crystal structure of the shank1 PDZ in complex with the βPIX C-terminal pentapeptide (642–646, DETNL) at 2.3 Å resolution and modeled shank1 PDZ binding to selected pentapeptide ligands. The resulting structures revealed a large hydrophobic pocket within the PDZ domain that can accommodate a variety of ligand residues at the P(0) position. A H-bond between His735 and Ser/Thr at the P(−2) position is invariant throughout the model structures. In addition, we identified multiple PDZ domain residues that are able to form H-bonds and salt bridges with an incoming target protein. Overall, our study provides a new level of understanding of the specificity and structural plasticity of the shank PDZ domain.

An Approach to Rational Ligand-Design Based on a Thermodynamic Analysis

Thermodynamic analysis is an effective tool in screening of lead-compounds for development of potential drug candidates. In most cases, a ligand achieve high affinity and specificity to a target protein by means of both favorable enthalpy and entropy terms, which can be reflected in binding profiles of Isothermal Titration Calorimetry (ITC). A favorable enthalpy change suggests the contribution of noncovalent contacts such as hydrogen bonding and van der Waals interaction between a ligand and its target protein. In general, optimization of binding enthalpy is more difficult than that of entropies in ligand-design; therefore, it is desirable to choose firstly a lead-compound based on its binding enthalpic gain. In this paper, we demonstrate the utility of thermodynamic approach to ligand screening using anti-ciguatoxin antibody 10C9 as a model of a target protein which possesses a large hydrophobic pocket. As a result of this screening, we have identified three compounds that could bind to the antigen-binding pocket of 10C9 with a few kcal/mol of favorable binding enthalpy. Comparison of their structure with the proper antigen ciguatoxin CTX3C revealed that 10C9 rigorously identifies their cyclic structure and a characteristic hydroxyl group. ITC measurement might be useful and powerful for a rational ligand screening and the optimization of the ligand; the enthalpic gain is an effective index for ligand-design studies.

Der p 5 Crystal Structure Provides Insight into the Group 5 Dust Mite Allergens

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of approximately 3000 A(3) that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

Structural Basis of the Recognition of the SAMP Motif of Adenomatous Polyposis Coli by the Src-Homology 3 Domain

Elucidation of the basis of interactions between biological molecules is essential for the understanding of living systems. Src-homology 3 (SH3) domains play critical roles in interaction networks of proteins by recognizing a proline-rich sequence motif, PxxP. There are, however, several SH3 domains that specifically bind to polypeptide chains without the conventional recognition sequence. The SH3 domain of DDEF1 associates with the SAMP motifs of the adenomatous polyposis coli (APC) tumor suppressor. The SAMP motifs are indispensable for the normal function of APC in tumor suppression. Here we present the structural basis of the interaction between the DDEF1-SH3 domain and the APC-SAMP motifs. We determined the solution structures of the DDEF1-SH3 domain both in a free state and in a complex with APC-SAMP. As the affinity of the interaction was not sufficiently high for the determination of the complex structure in solution by conventional methods, we utilized a fusion protein of the DDEF1-SH3 domain and APC-SAMP. The structures revealed that the SAMP motif adopts a class II polyproline type II helix even though it does not contain the PxxP motif and that a characteristically large hydrophobic pocket of the SH3 domain confers high selectivity to the interaction. Furthermore, investigation into the backbone dynamics of the free and bound systems by NMR spin relaxation experiments demonstrated that the DDEF1-SH3 domain exhibits high flexibility at the peptide recognition site in the absence of the ligand and that most residues of the APC-SAMP motif display extensive local motions even in the stable complex.

Crystal structure of the three FK506 binding protein domains of wheat FKBP73: evidence for a unique wFK73_2 domain

Here we describe the crystal structure of the N-terminal domain of the FK506-binding protein (FKBP) from wheat (wFKBP73), which is the first structure presenting three FK domains (wFK73_1, wFK73_2 and wFK73_3). The crystal model includes wFK73_2 and wFK73_3 domains and only part of the wFK73_1 domain. The wFK73_1 domain is responsible for binding FK506 and for peptidyl prolyl cis/trans isomerase (PPIase) activity, while the wFK73_2 and wFK73_3 domains lack these activities. A structure-based sequence comparison demonstrated that the absence of a large enough hydrophobic pocket important for PPIase activity, and of the conserved residues necessary for drug binding in the wFK73_2 and wFK73_3 domains explains the lack of these activities in these domains. Sequence and structural comparison between the three wFKBP73 domains suggest that the wFK73_2 domain is the most divergent. A structural comparison of the FK domains of wFKBP73 with other FKBPs containing more than one FK domain, revealed that while the overall architecture of each of the three FK domains displays a typical FKBP fold, their relative arrangement in space is unique and may have important functional implications. We suggest that the existence of FKBPs with three FK domains offers additional interactive options for these plant proteins enlarging the overall regulatory functions of these proteins.

Crystal Structure of a Full-Length Autotransporter

The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the β-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the β-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 Å. EstA has a relatively narrow, 12-stranded β-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the β-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by α-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.

χ-Conopeptide Pharmacophore Development: Toward a Novel Class of Norepinephrine Transporter Inhibitor (Xen2174) for Pain

Norepinephrine (NE) amplifies the strength of descending pain inhibition, giving inhibitors of spinal NET clinical utility in the management of pain. chi-MrIA isolated from the venom of a predatory marine snail noncompetitively inhibits NET and reverses allodynia in rat models of neuropathic pain. An analogue of chi-MrIA has been found to be a suitable drug candidate. On the basis of the NMR solution structure of this related peptide, Xen2174 (3), and structure-activity relationships of analogues, a pharmacophore model for the allosteric binding of 3 to NET is proposed. It is shown that 3 interacts with NET predominantly through amino acids in the first loop, forming a tight inverse turn presenting amino acids Tyr7, Lys8, and Leu9 in an orientation allowing for high affinity interaction with NET. The second loop interacts with a large hydrophobic pocket within the transporter. Analogues based on the pharmacophore demonstrated activities that support the proposed model. On the basis of improved chemical stability and a wide therapeutic index, 3 was selected for further development and is currently in phase II clinical trials.

Total synthesis and evaluation of 22-(3-azidobenzoyloxy)methyl epothilone C for photoaffinity labeling of β-tubulin

The total synthesis of 22-(3-azidobenzoyloxy)methyl epothilone C is described as a potential photoaffinity probe to elucidate the β-tubulin binding site. A sequential Suzuki-aldol-Yamaguchi macrolactonization strategy was utilized employing a novel derivatized C1–C6 fragment. The C22-functionalized analog exhibited good activity in microtubule assembly assays, but cytotoxicity was significantly reduced. Molecular modeling simulations indicated that excessive steric bulk in the C22 position is accommodated by the large hydrophobic pocket of the binding site. Photoaffinity labeling studies were inconclusive suggesting non-specific labeling.

The structural basis of lipopolysaccharide recognition by the TLR4–MD-2 complex

The lipopolysaccharide (LPS) of Gram negative bacteria is a well-known inducer of the innate immune response. Toll-like receptor (TLR) 4 and myeloid differentiation factor 2 (MD-2) form a heterodimer that recognizes a common 'pattern' in structurally diverse LPS molecules. To understand the ligand specificity and receptor activation mechanism of the TLR4-MD-2-LPS complex we determined its crystal structure. LPS binding induced the formation of an m-shaped receptor multimer composed of two copies of the TLR4-MD-2-LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD-2 and directly bridges the two components of the multimer. Five of the six lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD-2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD-2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophilic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD-2 indicates that two other lipid chains in LPS displace the phosphorylated glucosamine backbone by approximately 5 A towards the solvent area. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD-2. The TLR4-MD-2-LPS structure illustrates the remarkable versatility of the ligand recognition mechanisms employed by the TLR family, which is essential for defence against diverse microbial infection.

The IsdC Protein from Staphylococcus aureus Uses a Flexible Binding Pocket to Capture Heme

Staphylococcus aureus scavenges heme-iron from host hemoproteins using iron-regulated surface determinant (Isd) proteins. IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a NEAr Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX). The backbone coordinates of the ensemble of conformers representing the structure exhibit a root mean square deviation to the mean structure of 0.53 +/- 0.11 angstroms. IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its beta-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. Consistent with the structure and its role in heme transfer across the cell wall, we show that IsdC weakly binds heme (K(D) = 0.34 +/- 0.12 microm) and that ZnPPIX rapidly dissociates from the protein at a rate of 126 +/- 30 s(-1). NMR studies of the apo-form of IsdC reveal that a 3(10) helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins.

Crystal-Structure and Biochemical Characterization of Recombinant Human Calcyphosine Delineates a Novel EF-Hand-Containing Protein Family

Calcyphosine is an EF-hand protein involved in both Ca 2 + -phosphatidylinositol and cyclic AMP signal cascades, as well as in other cellular functions. The crystal structure of Ca 2 + -loaded calcyphosine was determined up to 2.65 Å resolution and reveals a protein containing two pairs of Ca 2 + -binding EF-hand motifs. Calcyphosine shares a highly similar overall topology with calmodulin. However, there are striking differences between EF-hand 4, both N-terminal and C-terminal regions, and interdomain linkers. The C-terminal domain of calcyphosine possesses a large hydrophobic pocket in the presence of calcium ions that might be implicated in ligand binding, while its N-terminal hydrophobic pocket is almost shielded by an additional terminal helix. Calcyphosine is largely monomeric, regardless of the presence of Ca 2 + . Differences in structure, oligomeric state in the presence and in the absence of Ca 2 + , a highly conserved sequence with low similarity to other proteins, and phylogeny define a new EF-hand-containing family of calcyphosine proteins that extends from arthropods to humans.

Determination of absolute configuration of secondary alcohols using lipase‐catalyzed kinetic resolutions

Lipases show high enantioselectivity toward a wide range of secondary alcohols. An empirical rule based on the relative sizes of the substituents predicts which enantiomer reacts faster. X-ray structures of lipases provide a molecular basis for this empirical rule: their alcohol-binding pocket contains large hydrophobic pocket open to solvent and another smaller pocket. This predictable enantiopreference of lipases allows the determination of the absolute configuration of secondary alcohols using lipase-catalyzed kinetic resolution. Researchers have used this relative method to determine the configuration of approximately 50 secondary alcohols either as the only method or in combination with other methods.

Structural Basis of Phospholipase Activity of Staphylococcus hyicus lipase

Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A 1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 Å resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A 1 activity.

Structure of Native Protein C Inhibitor Provides Insight into Its Multiple Functions

Protein C inhibitor (PCI) is a multifunctional serpin with wide ranging protease inhibitory functions, unique cofactor binding activities, and potential non-inhibitory functions akin to the hormone-transporting serpins. To gain insight into the molecular mechanisms utilized by PCI we developed a robust expression system in Escherichia coli and solved the crystal structure of PCI in its native state. The five monomers obtained from our two crystal forms provide an NMR-like ensemble revealing regions of inherent flexibility. The reactive center loop (RCL) of PCI is long and highly flexible with no evidence of hinge region incorporation into beta-sheet A, as seen for other heparin-binding serpins. We adapted an extrinsic fluorescence method for determining dissociation constants for heparin and find that the N-terminal tail of PCI and residues adjacent to helix H are not involved in heparin binding. The minimal heparin length capable of tight binding to PCI was determined to be chains of eight monosaccharide units. A large hydrophobic pocket occupied by hydrophobic crystal contacts was found in an analogous position to the hormone-binding site in thyroxine-binding globulin. In conclusion, the data presented here provide important insights into the mechanisms by which PCI exercises its multiple inhibitory and non-inhibitory functions.

Molecular Insights into Quorum Sensing in the Human Pathogen Pseudomonas aeruginosa from the Structure of the Virulence Regulator LasR Bound to Its Autoinducer

Many Gram-negative bacteria communicate via molecules called autoinducers to coordinate the activities of their populations. Such communication is termed quorum sensing and can regulate pathogenic virulence factor production and antimicrobial resistance. The quorum sensing system of Pseudomonas aeruginosa is currently the most intensively researched, because this bacterium is an opportunistic human pathogen annually responsible for the death of thousands of cystic fibrosis sufferers and many other immunocompromised individuals. Quorum sensing inhibitors can attenuate the pathogenicity of P. aeruginosa. Here we present the crystal structure of the P. aeruginosa LasR ligand-binding domain bound to its autoinducer 3-oxo-C(12)-acylhomoserine lactone. The structure is a symmetrical dimer, with each monomer exhibiting an alpha-beta-alpha fold similar to the TraR and SdiA quorum sensing proteins of Agrobacterium tumefaciens and Escherichia coli. The structure was determined up to 1.8-A resolution and reveals the atomic interactions between LasR and its autoinducer. The monomer structures of LasR, TraR, and SdiA are comparable but display differences in their quaternary organization. Inspection of their binding sites shows some unexpected variations resulting in quite different conformations of their bound autoinducers. We modeled interactions between LasR and various quorum sensing inhibitors, yielding insight into their possible mechanisms of action. The structure also provides a platform for the optimization, or de novo design, of quorum sensing inhibitors.

Structural basis for viral late-domain binding to Alix

The modular protein Alix is a central node in endosomal-lysosomal trafficking and the budding of human immunodeficiency virus (HIV)-1. The Gag p6 protein of HIV-1 contains a LYPx(n)LxxL motif that is required for Alix-mediated budding and binds a region of Alix spanning residues 360-702. The structure of this fragment of Alix has the shape of the letter 'V' and is termed the V domain. The V domain has a topologically complex arrangement of 11 alpha-helices, with connecting loops that cross three times between the two arms of the V. The conserved residue Phe676 is at the center of a large hydrophobic pocket and is crucial for binding to a peptide model of HIV-1 p6. Overexpression of the V domain inhibits HIV-1 release from cells. This inhibition of release is reversed by mutations that block binding of the Alix V domain to p6.

Structural determinants of the enantioselectivity of the hydroxynitrile lyase from Hevea brasiliensis

The hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis (HbHNL) is utilized as a biocatalyst in stereospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones. The catalyzed reaction represents one of the few industrially relevant examples of enzyme mediated C-C coupling reactions. In this work, we determined the X-ray crystal structures (at 1.54 and 1.76 Angstroms resolution) of HbHNL complexes with two chiral substrates -- mandelonitrile and 2,3-dimethyl-2-hydroxy-butyronitrile -- by soaking and rapid freeze quenching techniques. This is the first structural observation of the complex between a HNL and chiral substrates. Consistent with the known selectivity of the enzyme, only the S-enantiomers of the two substrates were observed in the active site. The binding modes of the chiral substrates were identical to that observed for the biological substrate acetone cyanohydrin. This indicates that the transformation of these non-natural substrates follows the same mechanism. A large hydrophobic pocket was identified in the active site of HbHNL which accommodates the more voluminous substituents of the two substrates. A three-point binding mode of the substrates -- hydrophobic pocket, hydrogen bonds between the hydroxyl group and Ser80 and Thr11, electrostatic interaction of the cyano group with Lys236 -- offers a likely structural explanation for the enantioselectivity of the enzyme. The structural data rationalize the observed (S)-enantioselectivity and form the basis for modifying the stereospecificity through rational design. The structures also revealed the necessity of considerable flexibility of the sidechain of Trp128 in order to bind and transform larger substrates.

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem-iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem-binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter-like beta-sandwich fold with a large hydrophobic haem-binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co-ordinated solely by Tyr(166). The phenol groups of Tyr(166) and Tyr(170) form an H-bond that may function in regulating haem binding and release. An analysis of IsdA structure-sequence alignments indicate that conservation of Tyr(166) is a predictor of haem binding by NEAT domains.