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HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.

Selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent (Ki = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Stichodactyla helianthus. Active helianthamide was produced in Escherichia coli via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitory motif. Helianthamide represents the first of a novel class of glycosidase inhibitors and provides an unusual example of functional malleability of the β-defensin fold, which is rarely seen outside of its traditional role in antimicrobial peptides., We describe the discovery and structural characterization of helianthamide, a peptide with highly potent and selective inhibitory activity against human pancreatic α-amylase.

The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233. This novel inhibitory motif can be reproduced in a greatly simplified analog, offering potential for new strategies for glycosidase inhibition and therapeutic development.

Cathepsin K (CatK) is the predominant mammalian bone-degrading protease and thus an ideal target for antiosteoporotic drug development. Rodent models of osteoporosis are preferred due to their close reflection of the human disease and their ease of handling, genetic manipulation and economic affordability. However, large differences in the potency of CatK inhibitors for the mouse/rat vs. the human protease orthologs have made it impossible to use rodent models. This is even more of a problem considering that the most advanced CatK inhibitors, including odanacatib (ODN) and balicatib, failed in human clinical trials due to side effects and rodent models are not available to investigate the mechanism of these failures. Here, we elucidated the structural elements of the potency differences between mouse and human CatK (hCatK) using ODN. We determined and compared the structures of inhibitor-free mouse CatK (mCatK), hCatK and ODN bound to hCatK. Two structural differences were identified and investigated by mutational analysis. Humanizing subsite 2 in mCatK led to a 5-fold improvement of ODN binding, whereas the replacement of Tyr61 in mCatK with Asp resulted in an hCatK with comparable ODN potency. Combining both sites further improved the inhibition of the mCatK variant. Similar results were obtained for balicatib. These findings will allow the generation of transgenic CatK mice that will facilitate the evaluation of CatK inhibitor adverse effects and to explore routes to avoid them.

The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

As part of a search for selective, mechanism-based covalent inhibitors of human pancreatic α-amylase we describe the chemoenzymatic synthesis of the disaccharide analog α-glucosyl epi-cyclophellitol, demonstrate its stoichiometric reaction with human pancreatic α-amylase and evaluate the time dependence of its inhibition. X-ray crystallographic analysis of the covalent derivative so formed confirms its reaction at the active site with formation of a covalent bond to the catalytic nucleophile D197. The structure illuminates the interactions with the active site and confirms OH4' on the nonreducing end sugar as a good site for attachment of fluorescent tags in generating probes for localization and quantitation of amylase in vivo.

The Syd protein has been implicated in the Sec-dependent transport of polypeptides across the bacterial inner membrane. Using Nanodiscs, we here provide direct evidence that Syd binds the SecY complex, and we demonstrate that interaction involves the two electropositive and cytosolic loops of the SecY subunit. We solve the crystal structure of Syd and together with cysteine cross-link analysis, we show that a conserved concave and electronegative groove constitutes the SecY-binding site. At the membrane, Syd decreases the activity of the translocon containing loosely associated SecY-SecE subunits, whereas in detergent solution Syd disrupts the SecYEG heterotrimeric associations. These results support the role of Syd in proofreading the SecY complex biogenesis and point to the electrostatic nature of the Sec channel interaction with its cytosolic partners.

Cathepsin K is the major collagenolytic protease in bone that facilitates physiological as well as pathological bone degradation. Despite its key role in bone remodeling and for being a highly sought-after drug target for the treatment of osteoporosis, the mechanism of collagen fiber degradation by cathepsin K remained elusive. Here, we report the structure of a collagenolytically active cathepsin K protein dimer. Cathepsin K is organized into elongated C-shaped protease dimers that reveal a putative collagen-binding interface aided by glycosaminoglycans. Molecular modeling of collagen binding to the dimer indicates the participation of nonactive site amino acid residues, Q21 and Q92, in collagen unfolding. Mutations at these sites as well as perturbation of the dimer protein–protein interface completely inhibit cathepsin-K–mediated fiber degradation without affecting the hydrolysis of gelatin or synthetic peptide. Using scanning electron microscopy, we demonstrate the specific binding of cathepsin K at the edge of the fibrillar gap region of collagen fibers, which suggest initial cleavage events at the N - and C -terminal ends of tropocollagen molecules. Edman degradation analysis of collagen fiber degradation products revealed those initial cleavage sites. We propose that one cathepsin K molecule binds to collagen-bound glycosaminoglycans at the gap region and recruits a second protease molecule that provides an unfolding and cleavage mechanism for triple helical collagen. Removal of collagen-associated glycosaminoglycans prevents cathepsin K binding and subsequently fiber hydrolysis. Cathepsin K dimer and glycosaminoglycan binding sites represent novel targeting sites for the development of nonactive site-directed second-generation inhibitors of this important drug target.

The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.

A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1 H NMR spectroscopy indicates that Mn 2+ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn 2+ is 1.28(4) × 10 4 M −1 (pH 6.96, ionic strength I = 17.2 μM, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pK a for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn 2+ -peroxidase activity of the variant are both increased ≈3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn 2+ . The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn 2+ and Cd 2+ . Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation.

Autotransporters (ATs) are proteins that deliver effectors (the passenger domain) to the surface of Gram-negative bacteria by the type V secretion pathway. The passenger domain of BrkA, a Bordetella pertussis autotransporter mediating serum resistance and adherence, was cloned in a pET expression system and overexpressed in Escherichia coli. The gene product was correctly refolded, purified to homogeneity and crystallized. The crystals diffracted to 2.8 A resolution. The space group was assumed to be P4(1)2(1)2, with unit-cell parameters a = b = 108.19, c = 115.35 A.

Study of the hexameric and allosterically regulated citrate synthases (type II CS) provides a rare opportunity to gain not only an understanding of a novel allosteric mechanism but also insight into how such properties can evolve from an unregulated structural platform (the dimeric type I CS). To address both of these issues, we have determined the structure of the complex of NADH (a negative allosteric effector) with the F383A variant of type II Escherichia coli CS. This variant was chosen because its kinetics indicate it is primarily in the T or inactive allosteric conformation, the state that strongly binds to NADH. Our structural analyses show that the six NADH binding sites in the hexameric CS complex are located at the interfaces between dimer units such that most of each site is formed by one subunit, but a number of key residues are drawn from the adjacent dimer. This arrangement of interactions serves to explain why NADH allosteric regulation is a feature only of hexameric type II CS. Surprisingly, in both the wild-type enzyme and the NADH complex, the two subunits of each dimer within the hexameric conformation are similar but not identical in structure, and therefore, while the general characteristics of NADH binding interactions are similar in each subunit, the details of these are somewhat different between subunits. Detailed examination of the observed NADH binding sites indicates that both direct charged interactions and the overall cationic nature of the sites are likely responsible for the ability of these sites to discriminate between NADH and NAD(+). A particularly novel characteristic of the complex is the horseshoe conformation assumed by NADH, which is strikingly different from the extended conformation found in its complexes with most proteins. Sequence homology studies suggest that this approach to binding NADH may arise out of the evolutionary need to add an allosteric regulatory function to the base CS structure. Comparisons of the amino acid sequences of known type II CS enzymes, from different Gram-negative bacteria taxonomic groups, show that the NADH-binding residues identified in our structure are strongly conserved, while hexameric CS molecules that are insensitive to NADH have undergone key changes in the sequence of this part of the protein.

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose to trehalose in mycobacteria as one of three biosynthetic pathways to this non-reducing disaccharide. Given the importance of trehalose to survival of mycobacteria there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site cleft within which the intramolecular rearrangement can occur with minimal hydrolysis. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation.

Human heat shock protein 27 (Hsp27) is an oligomeric and cell survival protein that has been associated with several cancers including prostrate and breast cancer. It's a known anti-apoptotic protein that functions as a molecular chaperone for several proteins. Hsp27 characteristically binds and stabilizes numerous partially unfolded proteins preventing their degradation, and has been shown to prevent actin polymerization in vitro. Several actin-binding residues involved in this interaction have been identified at the N-terminal loop and highly conserved alpha crystallin domains of Hsp27. Multiple assays have demonstrated that this hydrophobic actin-binding site is also involved in other protein binding. We therefore propose a common substrate-binding region on Hsp27 and present a model of Hsp27 binding to actin.

We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.

The 1.8 A resolution structure of the glycosyl-enzyme intermediate formed on the retaining beta-1,4-xylanase from Bacillus circulans has been determined using X-ray crystallographic techniques. The 2-fluoro-xylose residue bound in the -1 subsite adopts a 2,5B (boat) conformation, allowing atoms C5, O5, C1, and C2 of the sugar to achieve coplanarity as required at the oxocarbenium ion-like transition states of the double-displacement catalytic mechanism. Comparison of this structure to that of a mutant of this same enzyme noncovalently complexed with xylotetraose [Wakarchuk et al. (1994) Protein Sci. 3, 467-475] reveals a number of differences beyond the distortion of the sugar moiety. Most notably, a bifurcated hydrogen bond interaction is formed in the glycosyl-enzyme intermediate involving Heta of Tyr69, the endocyclic oxygen (O5) of the xylose residue in the -1 subsite, and Oepsilon2 of the catalytic nucleophile, Glu78. To gain additional understanding of the role of Tyr69 at the active site of this enzyme, we also determined the 1.5 A resolution structure of the catalytically inactive Tyr69Phe mutant. Interestingly, no significant structural perturbation due to the loss of the phenolic group is observed. These results suggest that the interactions involving the phenolic group of Tyr69, O5 of the proximal saccharide, and Glu78 Oepsilon2 are important for the catalytic mechanism of this enzyme, and it is proposed that, through charge redistribution, these interactions serve to stabilize the oxocarbenium-like ion of the transition state. Studies of the covalent glycosyl-enzyme intermediate of this xylanase also provide insight into specificity, as contacts with C5 of the xylose moiety exclude sugars with hydroxymethyl substituents, and the mechanism of catalysis, including aspects of stereoelectronic theory as applied to glycoside hydrolysis.