Kinase inhibitors that bind to the ATP cleft can be broadly classified into two groups: those that bind exclusively to the ATP site with the kinase assuming a conformation otherwise conducive to ...phosphotransfer (type I), and those that exploit a hydrophobic site immediately adjacent to the ATP pocket made accessible by a conformational rearrangement of the activation loop (type II). To date, all type II inhibitors were discovered by using structure-activity-guided optimization strategies. Here, we describe a general pharmacophore model of type II inhibition that enables a rational “hybrid-design” approach whereby a 3-trifluoromethylbenzamide functionality is appended to four distinct type I scaffolds in order to convert them into their corresponding type II counterparts. We demonstrate that the designed compounds function as type II inhibitors by using biochemical and cellular kinase assays and by cocrystallography with Abl.
Factor XIII-cross-linked fragment D (double-D) from human fibrin was crystallized in the presence of two different peptide ligands and the X-ray structure determined at 2.3 Å. The peptide ...Gly-Pro-Arg-Pro-amide, which is an analogue of the knob exposed by the thrombin-catalyzed removal of fibrinopeptide A, was found to reside in the γ-chain holes, and the peptide Gly-His-Arg-Pro-amide, which corresponds to the β-chain knob, was found in the homologous β-chain holes. The structure shows for the first time that the β-chain knob does indeed bind to a homologous hole on the β-chain. The γ- and β-chain holes are structurally very similar, and it is remarkable they are able to distinguish between these two peptides that differ by a single amino acid. Additionally, we have found that the β-chain domain, like its γ-chain counterpart, binds calcium.
A series of
N
α-2-benzoxazolyl-α-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure–activity relationships including ...strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound
3 bound to the active site of cathepsin S is also reported.
A series of
N
α-2-benzoxazolyl-α-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure–activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound
3 bound to the active site of cathepsin S is also reported.
It has been recently shown that orthogonal tRNA/aminoacyl-tRNA synthetase pairs can be evolved to allow genetic incorporation of unnatural amino acids into proteins in both prokaryotes and ...eukaryotes. Here we describe the crystal structure of an evolved aminoacyl-tRNA synthetase that charges the unnatural amino acid p-acetylphenylalanine. Molecular recognition is due to altered hydrogen bonding and packing interactions with bound substrate that result from changes in both side-chain and backbone conformation.
The structure of two Thermotoga maritima proteins, a conserved hypothetical protein (TM0160) and a transcriptional regulator (TM1171), have now been determined at 1.9 Å and 2.3 Å resolution, ...respectively, as part of a large‐scale structural genomics project. Our first efforts to crystallize full‐length versions of these targets were unsuccessful. However, analysis of the recombinant purified proteins using the technique of enhanced amide hydrogen/deuterium exchange mass spectroscopy (DXMS) revealed substantial regions of rapid amide deuterium hydrogen exchange, consistent with flexible regions of the structures. Based on these exchange data, truncations were designed to selectively remove the disordered C‐terminal regions, and the resulting daughter proteins showed greatly enhanced crystallizability. Comparative DXMS analysis of full‐length protein versus truncated forms demonstrated complete and exact preservation of the exchange rate profiles in the retained sequence, indicative of conservation of the native folded structure. This study presents the first structures produced with the aid of the DXMS method for salvaging intractable crystallization targets. The structure of TM0160 represents a new fold and highlights the use of this approach where any prior structural knowledge is absent. The structure of TM1171 represents an example where the lack of a substrate/cofactor may impair crystallization. The details of both structures are presented and discussed.
Structure-based design was utilized to guide the early stage optimization of a substrate-like inhibitor to afford potent peptidomimetic inhibitors of the channel-activating protease prostasin. The ...first X-ray structure of a small molecule inhibitor bound to the active site of prostasin is also reported.
Structure-based design was utilized to guide the early stage optimization of a substrate-like inhibitor to afford potent peptidomimetic inhibitors of the channel-activating protease prostasin. The first X-ray crystal structures of prostasin with small molecule inhibitors bound to the active site are also reported.
Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) are strongly associated with levels of low-density lipoprotein cholesterol in the blood plasma and, thereby, occurrence or ...resistance to atherosclerosis and coronary heart disease. Despite this importance, relatively little is known about the biology of PCSK9. Here, the crystal structure of a full-length construct of PCSK9 solved to 1.9-Aa resolution is presented. The structure contains a fully folded C-terminal cysteine-rich domain (CRD), showing a distinct structural similarity to the resistin homotrimer, a small cytokine associated with obesity and diabetes. This structural relationship between the CRD of PCSK9 and the resistin family is not observed in primary sequence comparisons and strongly suggests a distant evolutionary link between the two molecules. This three-dimensional homology provides insight into the function of PCSK9 at the molecular level and will help to dissect the link between PCSK9 and CHD.
AvrB is a
Pseudomonas syringae type III effector protein that is translocated into host plant cells during attempted pathogenesis.
Arabidopsis harboring the corresponding resistance protein RPM1 can ...detect AvrB and mount a rapid host defense response, thus avoiding active infection. In the plant cell, AvrB induces phosphorylation of RIN4, a key component in AvrB/RPM1 recognition. Although the AvrB/RPM1 system is among the best characterized of the numerous bacterial effector/plant resistance protein systems involved in plant disease resistance and pathogenesis, the details of the molecular recognition mechanism are still unclear. To gain further insights, the crystal structure of AvrB was determined. The 2.2 Å structure exhibits a novel mixed α/β bilobal fold. Aided by the structural information, we demonstrate that one lobe is the determinant of AvrB/RPM1 recognition specificity. This structural information and preliminary structure-function studies provide a framework for the future understanding of AvrB function on the molecular level.
An algorithm is described which utilizes the solvent mask generated by the solvent‐flattening technique to calculate a monomer molecular envelope. In the case where non‐crystallographic symmetry ...(NCS) is present in the crystal and self‐rotation angles are known from a self‐rotation function, the resultant monomer envelopes can be used to search for the translation component of the NCS element by a three‐dimensional search in real space. In the absence of self‐rotation angles, the monomer envelope may be used to derive the NCS operators by reciprocal‐space techniques. Thus, an automatic procedure for averaging directly from the solvent‐flattening stage can be implemented. The procedure was instrumental in the structure solution of fibrinogen fragment D, which is presented as an example.
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