Thex-raycrystal structure ofapeptide designed toform adouble-stranded parallel coiled coil showsthat itisactually atriple-stranded coiled coil formed bythree a-helices. Unlike thedesigned parallel coiled coil, thehelices runup-up-down. Thestructure isstabilized by adistinctive hydrophobic interface consisting ofeight layers. Asinthedesign, eacha-helix inthecoiled coil contributes oneleucine sidechain toeachlayer. Thestructure suggests that hydrophobic interactions areadominant factor inthestabilization ofcoiled coils. The stoichiometry andgeometry ofcoiled coils areprimarily determined bysidechain packing inthesolvent-inaccessible interior, butelectrostatic interactions alsocontribute.

Inhibition of the biosynthesis of proinflammatory cytokines such as tumor necrosis factor and interleukin-1 via p38 has been an approach toward the development of a disease modifying agent for the treatment of chronic inflammation and autoimmune diseases. The development of a new core structure of p38 inhibitors, 3-(4-fluorophenyl)-2-(pyridin-4-yl)-1H-pyrrolo[3,2-b] pyridine, is described. X-ray crystallographic data of the lead bound to the active site of p38 was used to guide the optimization of the series. Specific focus was placed on modulating the physical properties of the core while maintaining potent inhibition of p38. These efforts identified 42c as a potent inhibitor of p38, which also possessed the required physical properties worthy of advanced studies.

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

Cdc25 phosphatases activate the cell division kinases throughout the cell cycle. The 2.3 A structure of the human Cdc25A catalytic domain reveals a small alpha/beta domain with a fold unlike previously described phosphatase structures but identical to rhodanese, a sulfur-transfer protein. Only the active-site loop, containing the Cys-(X)5-Arg motif, shows similarity to the tyrosine phosphatases. In some crystals, the catalytic Cys-430 forms a disulfide bond with the invariant Cys-384, suggesting that Cdc25 may be self-inhibited during oxidative stress. Asp-383, previously proposed to be the general acid, instead serves a structural role, forming a conserved buried salt-bridge. We propose that Glu-431 may act as a general acid. Structure-based alignments suggest that the noncatalytic domain of the MAP kinase phosphatases will share this topology, as will ACR2, a eukaryotic arsenical resistance protein.

BACKGROUND:The design of amino acid sequences that adopt a desired three-dimensional fold has been of keen interest over the past decade. However, the design of proteins that adopt unique conformations is still a considerable problem. Until very recently, all of the designed proteins that have been extensively characterized possess the hallmarks of the molten globular state. Molten globular intermediates have been observed in both equilibrium and kinetic protein folding/stability studies, and understanding the forces that determine compact non-native states is critical for a comprehensive understanding of proteins. This paper describes the solution and early solid state characterization of peptides that form molten globular ensembles.RESULTS & CONCLUSIONS:Crystals diffracting to 3.5 A resolution have been grown of a 16-residue peptide (alpha 1A) designed to form a tetramer of alpha-helices. In addition, a closely related peptide, alpha 1, has previously been shown to yield crystals that diffract to 1.2 A resolution. The solution properties of these two peptides were examined to determine whether their well defined crystalline conformations were retained in solution. On the basis of an examination of their NMR spectra, sedimentation equilibria, thermal unfolding, and ANS binding, it is concluded that the peptides form alpha-helical aggregates with properties similar to those of the molten globule state. Thus, for these peptides, the process of crystallization bears many similarities to models of protein folding. Upon dissolution, the peptides rapidly assume compact molten globular states similar to the molten globule like intermediates that are formed at short times after refolding is initiated. Following a rate-determining nucleation step, the peptides crystallize into a single or a small number of conformations in a process that mimics the formation of native structure in proteins.

Annals of the New York Academy of SciencesVolume 732, Issue 1 p. 375-378 Structural Analysis of the Catalytic Domain of Human Fibroblast Collagenase BRETT LOVEJOY, BRETT LOVEJOY Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorANNE CLEASBY, ANNE CLEASBY Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorANNE M. HASSELL, ANNE M. HASSELL Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorMICHAEL A. LUTHER, MICHAEL A. LUTHER Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorDEBRA WEIGL, DEBRA WEIGL Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorGERARD McGEEHAN, GERARD McGEEHAN Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorMILLARD H. LAMBERT, MILLARD H. LAMBERT Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorSTEVEN R. JORDAN, STEVEN R. JORDAN Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this author BRETT LOVEJOY, BRETT LOVEJOY Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorANNE CLEASBY, ANNE CLEASBY Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorANNE M. HASSELL, ANNE M. HASSELL Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorMICHAEL A. LUTHER, MICHAEL A. LUTHER Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorDEBRA WEIGL, DEBRA WEIGL Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorGERARD McGEEHAN, GERARD McGEEHAN Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorMILLARD H. LAMBERT, MILLARD H. LAMBERT Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this authorSTEVEN R. JORDAN, STEVEN R. JORDAN Glaxo Research Institute 5 Moore Drive Research Triangle Park, North Carolina 27709Search for more papers by this author First published: September 1994 https://doi.org/10.1111/j.1749-6632.1994.tb24758.xCitations: 11AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume732, Issue1Inhibition of Matrix Metalloproteinases: Therapeutic PotentialSeptember 1994Pages 375-378 RelatedInformation

Collagenase is a member of the matrix metalloproteinase (MMP) family of enzymes. Aberrant regulation of this family has been implicated in pathologies such as arthritis and metastasis. Two crystal forms of the catalytic (19-kDa) domain of human fibroblast collagenase have been determined using collagenase complexed with a peptide-based inhibitor (CPLX) as a starting model [Lovejoy et al. (1994) Science 263, 375]. The first crystal form (CF1) contains one molecule in the asymmetric unit and has been determined at 1.9-A resolution with an R factor of 19.8%. The second crystal form (CF2) contains two molecules (A and B) in the asymmetric unit and has been determined at 2.1-A resolution with an R factor of 19.7%. The catalytic domain of collagenase is spherical with an active site cleft that contains a ligated catalytic zinc ion. Collagenase shares some structural homology with the bacterial zinc proteinase, thermolysin [Matthews et al. (1972) Nature, New Biol. 238, 37], and the crayfish digestive peptidase, astacin [Bode et al. (1992) Nature 358, 164]. The amino terminus (Leu 102 to Gly 105) of CF1 and CF2 molecules A and B differs from the conformation found in CPLX by bending away from the molecule and interacting with the active site cleft of symmetry-related molecules. In this alternative conformation, both the mainchain nitrogen and carbonyl oxygen of Leu 102 ligate the symmetry-related catalytic zinc. Although there are structural differences in the active site clefts of CF1, CF2, and CPLX, a number of complex-stabilizing interactions are conserved. The structure of collagenase will be useful for developing compounds that selectively inhibit individual members of the closely related matrix metalloproteinase family.

Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.

The x-ray crystal structure of a peptide designed to form a double-stranded parallel coiled coil shows that it is actually a triple-stranded coiled coil formed by three α-helices. Unlike the designed parallel coiled coil, the helices run up-up-down. The structure is stabilized by a distinctive hydrophobic interface consisting of eight layers. As in the design, each α-helix in the coiled coil contributes one leucine side chain to each layer. The structure suggests that hydrophobic interactions are a dominant factor in the stabilization of coiled coils. The stoichiometry and geometry of coiled coils are primarily determined by side chain packing in the solvent-inaccessible interior, but electrostatic interactions also contribute.

The crystal structures of recombinant canine and bovine granulocyte colony stimulating factor (G-CSF) have been determined by X-ray crystallography, using molecular replacement with recombinant human G-CSF as a model. G-CSF is a member of the cytokine family of glycoproteins that stimulate the differentiation and proliferation of blood cells. Human, bovine and canine G-CSF all have a molecular mass of about 19 kDa and share an amino acid sequence identity of about 80%. Two crystal forms of canine G-CSF have been solved. Form I recombinant canine G-CSF (rcG-CSFI; space group C2) contains one molecule in the asymmetric unit while form II canine G-CSF (rcG-CSFII; space group P21) has two molecules in the asymmetric unit and bovine G-CSF (rbG-CSF; space group P 212121) contains one molecule in the asymmetric unit. rcG-CSFI has been refined to an R factor of 20·7% with data to 2·3 Å resolution and rcG-CSFII has been refined to an R factor of 19·3% with data to 2·2 Å resolution. rbG-CSF has been refined to an R factor of 21·3% with data to 1·7 Å resolution. The structure of human, canine and bovine G-CSF is an antiparallel 4-α-helical bundle with up-up-down-down connectivity. With the exception of one highly exposed loop (residues 66 to 74), the human, canine and bovine structures are very similar to each other. Using our series of G-CSF crystal structures we developed a function that describes the probability that a particular residue position (i) contributes to a G-CSF receptor binding site based on two principles, (1) high sequence conservation in the primary sequence of human, bovine, canine and murine G-CSF and (2) conservation of high solvent accessibility in the human, bovine and canine crystal structures. On the basis of this probability function as well as a comparison of G-CSF to the crystal structure of human growth hormone (hGH) complexed with the extracellular domain of the human growth hormone receptor (hGHbp), residues that contribute to potential G-CSF receptor binding sites are identified.