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Biomedical subjects

R A Engh

Publications and source records attributed to R A Engh.

At least 19 recordsLinked to original sources
PubMed2001

High thermostability and lack of cooperative DNA binding distinguish the p63 core domain from the homologous tumor suppressor p53.

The p53 protein is the major tumor suppressor in mammals. The discovery of the p53 homologs p63 and p73 defined a family of p53 members with distinct roles in tumor suppression, differentiation, and development. Here, we describe the biochemical characterization of the core DNA-binding domain of a human isoform of p63, p63-delta, and particularly novel features in comparison with p53. In contrast to p53, the free p63 core domain did not show specific binding to p53 DNA consensus sites. However, glutathione S-transferase-fused and thus dimerized p63 and p53 core domains had similar affinity and specificity for the p53 consensus sites p21, gadd45, cyclin G, and bax. Furthermore, the fold of p63 core was remarkably stable compared with p53 as judged by differential scanning calorimetry (T(m) = 61 degrees C versus 44 degrees C for p53) and equilibrium unfolding ([urea](50%) = 5.2 m versus 3.1 m for p53). A homology model of p63 core highlights differences at a segment near the H1 helix hypothetically involved in the formation of the dimerization interface in p53, which might reduce cooperativity of p63 core DNA binding compared with p53. The model also shows differences in the electrostatic and hydrophobic potentials of the domains relevant to folding stability.

Amino Acid Sequence↗
PubMed2001

The interaction of insulin-like growth factor-I with the N-terminal domain of IGFBP-5.

Insulin-like growth factors (IGFs) are key regulators of cell proliferation, differentiation and transformation, and are thus pivotal in cancer, especially breast, prostate and colon neoplasms. They are also important in many neurological and bone disorders. Their potent mitogenic and anti-apoptotic actions depend primarily on their availability to bind to the cell surface IGF-I receptor. In circulation and interstitial fluids, IGFs are largely unavailable as they are tightly associated with IGF-binding proteins (IGFBPs) and are released after IGFBP proteolysis. Here we report the 2.1 A crystal structure of the complex of IGF-I bound to the N-terminal IGF-binding domain of IGFBP-5 (mini-IGFBP-5), a prototype interaction for all N-terminal domains of the IGFBP family. The principal interactions in the complex comprise interlaced hydrophobic side chains that protrude from both IGF-I and the IGFBP-5 fragment and a surrounding network of polar interactions. A solvent-exposed hydrophobic patch is located on the IGF-I pole opposite to the mini-IGFBP-5 binding region and marks the IGF-I receptor binding site.

Amino Acid Sequence↗
PubMed2001

The extracellular human melanoma inhibitory activity (MIA) protein adopts an SH3 domain-like fold.

Melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma, as enhanced values diagnose metastatic melanoma stages III and IV. Here we show that the recombinant human MIA adopts an SH3 domain-like fold in solution, with two perpendicular, antiparallel, three- and five-stranded beta-sheets. In contrast to known structures with the SH3 domain fold, MIA is a single-domain protein, and contains an additional antiparallel beta-sheet and two disulfide bonds. MIA is also the first extracellular protein found to have the SH3 domain-like fold. Furthermore, we show that MIA interacts with fibronectin and that the peptide ligands identified for MIA exhibit a matching sequence to type III human fibronectin repeats, especially to FN14, which is close to an integrin alpha4beta1 binding site. The present study, therefore, may explain the role of MIA in metastasis in vivo, and supports a model in which the binding of human MIA to type III repeats of fibronectin competes with integrin binding, thus detaching cells from the extracellular matrix.

Amino Acid Sequence↗
PubMed2001

The 1.8-A crystal structure of a matrix metalloproteinase 8-barbiturate inhibitor complex reveals a previously unobserved mechanism for collagenase substrate recognition.

The individual zinc endoproteinases of the tissue degrading matrix metalloproteinase (MMP) family share a common catalytic architecture but are differentiated with respect to substrate specificity, localization, and activation. Variation in domain structure and more subtle structural differences control their characteristic specificity profiles for substrates from among four distinct classes (Nagase, H., and Woessner, J. F. J. (1999) J. Biol. Chem. 274, 21491-21494). Exploitation of these differences may be decisive for the design of anticancer or other drugs, which should be highly selective for their particular MMP targets. Based on the 1.8-A crystal structure of human neutrophil collagenase (MMP-8) in complex with an active site-directed inhibitor (RO200-1770), we identify and describe new structural determinants for substrate and inhibitor recognition in addition to the primary substrate recognition sites. RO200-1770 induces a major rearrangement at a position relevant to substrate recognition near the MMP-8 active site (Ala206-Asn218). In stromelysin (MMP-3), competing stabilizing interactions at the analogous segment hinder a similar rearrangement, consistent with kinetic profiling of several MMPs. Despite the apparent dissimilarity of the inhibitors, the central 2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor RO200-1770 mimics the interactions of the hydroxamate-derived inhibitor batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T., Pieper, M., Tschesche, H., Huber, R., and Bode, W. (1995) Eur. J. Biochem. 228, 830-841) for binding to MMP-8. The two additional phenyl and piperidyl ring substituents of the inhibitor bind into the S1' and S2' pockets of MMP-8, respectively. The crystal lattice contains a hydrogen bond between the O(gamma) group of Ser209 and N(delta)1 of His207 of a symmetry related molecule; this interaction suggests a model for recognition of hydroxyprolines present in physiological substrates. We also identify a collagenase-characteristic cis-peptide bond, Asn188-Tyr189, on a loop essential for collagenolytic activity. The sequence conservation pattern at this position marks this cis-peptide bond as a determinant for triple-helical collagen recognition and processing.

Amino Acid Sequence↗
PubMed2001

Chalcone derivatives antagonize interactions between the human oncoprotein MDM2 and p53.

The oncoprotein MDM2 inhibits the tumor suppressor protein p53 by binding to the p53 transactivation domain. The p53 gene is inactivated in many human tumors either by mutations or by binding to oncogenic proteins. In some tumors, such as soft tissue sarcomas, overexpression of MDM2 inactivates an otherwise intact p53, disabling the genome integrity checkpoint and allowing cell cycle progression of defective cells. Disruption of the MDM2/p53 interaction leads to increased p53 levels and restored p53 transcriptional activity, indicating restoration of the genome integrity check and therapeutic potential for MDM2/p53 binding antagonists. Here, we show by multidimensional NMR spectroscopy that chalcones (1,3-diphenyl-2-propen-1-ones) are MDM2 inhibitors that bind to a subsite of the p53 binding cleft of human MDM2. Biochemical experiments showed that these compounds can disrupt the MDM2/p53 protein complex, releasing p53 from both the p53/MDM2 and DNA-bound p53/MDM2 complexes. These results thus offer a starting basis for structure-based drug design of cancer therapeutics.

Antineoplastic Agents↗
PubMed1999

Coagulation factor IXa: the relaxed conformation of Tyr99 blocks substrate binding.

BACKGROUND: Among the S1 family of serine proteinases, the blood coagulation factor IXa (fIXa) is uniquely inefficient against synthetic peptide substrates. Mutagenesis studies show that a loop of residues at the S2-S4 substrate-binding cleft (the 99-loop) contributes to the low efficiency. The crystal structure of porcine fIXa in complex with the inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was unable to directly clarify the role of the 99-loop, as the doubly covalent inhibitor induced an active conformation of fIXa. RESULTS: The crystal structure of a recombinant two-domain construct of human fIXa in complex with p-aminobenzamidine shows that the Tyr99 sidechain adopts an atypical conformation in the absence of substrate interactions. In this conformation, the hydroxyl group occupies the volume corresponding to the mainchain of a canonically bound substrate P2 residue. To accommodate substrate binding, Tyr99 must adopt a higher energy conformation that creates the S2 pocket and restricts the S4 pocket, as in fIXa-PPACK. The energy cost may contribute significantly to the poor K(M) values of fIXa for chromogenic substrates. In homologs, such as factor Xa and tissue plasminogen activator, the different conformation of the 99-loop leaves Tyr99 in low-energy conformations in both bound and unbound states. CONCLUSIONS: Molecular recognition of substrates by fIXa seems to be determined by the action of the 99-loop on Tyr99. This is in contrast to other coagulation enzymes where, in general, the chemical nature of residue 99 determines molecular recognition in S2 and S3-S4. This dominant role on substrate interaction suggests that the 99-loop may be rearranged in the physiological fX activation complex of fIXa, fVIIIa, and fX.

Amino Acid Sequence↗
PubMed1999

Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius.

Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe here the 2.5 A resolution crystal structure of a DNA polymerase from the Archaea Thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function. Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA-protein interface. In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site. A physiological role of this "closed" conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents.

Amino Acid Sequence↗
PubMed1999

Comparison of two implementations of the incremental construction algorithm in flexible docking of thrombin inhibitors.

A set of 32 known thrombin inhibitors representing different chemical classes has been used to evaluate the performance of two implementations of incremental construction algorithms for flexible molecular docking: DOCK 4.0 and FlexX 1.5. Both docking tools are able to dock 10-35% of our test set within 2 A of their known, bound conformations using default sampling and scoring parameters. Although flexible docking with DOCK or FlexX is not able to reconstruct all native complexes, it does offer a significant improvement over rigid body docking of single, rule-based conformations, which is still often used for docking of large databases. Docking of sets of multiple conformers of each inhibitor, obtained with a novel protocol for diverse conformer generation and selection, yielded results comparable to those obtained by flexible docking. Chemical scoring, which is an empirically modified force field scoring method implemented in DOCK 4.0, outperforms both interaction energy scoring by DOCK and the Böhm scoring function used by FlexX in rigid and flexible docking of thrombin inhibitors. Our results indicate that for reliable docking of flexible ligands the selection of anchor fragments, conformational sampling and currently available scoring methods still require improvement.

Algorithms↗
PubMed1998

Novel acylguanidine containing thrombin inhibitors with reduced basicity at the P1 moiety.

Replacement of the noragmatine group in thrombin inhibitors with a beta-alanyl-guanidine group resulted in a nearly equipotent and more selective compound 8 despite the fact that the pKa of this P1 moiety is five orders of magnitude lower. Further modification resulted in a nonpeptide inhibitor with this beta-alanyl-guanidine group, compound 28. This is an active and selective thrombin inhibitor and in view of its nonpeptide/low basicity structure selected for further pharmacological studies.

Antithrombins↗
PubMed1998

Tetrahydro-isoquinoline-based factor Xa inhibitors.

Derivatives of (2-amidino-1,2,3, 4-tetrahydro-isoquinolin-7-yloxy)phenylacetic acid (TIPAC) were developed as inhibitors of factor Xa (fXa). The compounds are prepared using 15 synthetic steps on average. The most potent compounds (14, 17, 22-26) display inhibition constants of Ki = 21-55 nM but do not inhibit thrombin (Ki = 5->100 microM) and only weakly inhibit trypsin (Ki = 0.08-5 microM). They bear a second basic moiety, e.g., substituted 1-(iminomethyl)piperidines, which is linked to C-4 of the phenyl group of TIPAC via an oxygen atom. The inhibition constants of these compounds are almost independent of the size of the (iminomethyl)piperidine substituent. Due to the fact that fXa displays two cation binding sites, namely, the S1 and S4 sites, in principle two binding modes are conceivable for the novel dibasic fXa inhibitors. Molecular modeling experiments based on the X-ray structures of uninhibited fXa and the DX-9065a/fXa complex were carried out. The results taken together with the inhibition constants clearly favor one binding mode: the tetrahydro-isoquinoline fills the S1 pocket even better than the naphthalene moiety of DX-9065a, and the (iminomethyl)piperidine residues occupy the S4 site.

Anticoagulants↗
PubMed1998

Structure of human cyclin-dependent kinase inhibitor p19INK4d: comparison to known ankyrin-repeat-containing structures and implications for the dysfunction of tumor suppressor p16INK4a.

BACKGROUND: The four members of the INK4 gene family (p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d)) inhibit the closely related cyclin-dependent kinases CDK4 and CDK6 as part of the regulation of the G1-->S transition in the cell-division cycle. Loss of INK4 gene product function, particularly that of p16(INK4a), is found in 10-60% of human tumors, suggesting that broadly applicable anticancer therapies might be based on restoration of p16(INK4a) CDK inhibitory function. Although much less frequent, defects of p19(INK4d) have also been associated with human cancer (osteosarcomas). The protein structures of some INK4 family members, determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray techniques, have begun to clarify the functional role of p16(INK4a) and the dysfunction introduced by the mutations associated with human tumors. RESULTS: The crystal structure of human p19(INK4d) has been determined at 1.8 A resolution using multiple isomorphous replacement methods. The fold of p19(INK4d) produces an oblong molecule comprising five approximately 32-residue ankyrin-like repeats. The architecture of the protein demonstrates the high structural similarity within the INK4 family. Comparisons to other ankyrin-repeat-containing proteins (GABPbeta, 53BP2 and myotrophin) show similar structures with comparable hydrogen-bonding patterns and hydrophobic interactions. Such comparisons highlight the splayed beta-loop geometry that is specific to INK4 inhibitors. This geometry is the result of a modified ankyrin structure in the second repeat. CONCLUSIONS: Among the INK4 inhibitors, the highest amino acid sequence conservation is found in the helical stacks; this conservation creates a conserved beta-loop geometry specific to INK4 inhibitors. Therefore, in addition to models which predict that the conserved helix alpha6 is responsible for CDK inhibition, a binding mode whereby the loops of INK4 proteins bind to the CDKs should also be considered. A similar loop-based interaction is seen in the complex formed between the ankyrin-repeat-containing protein GABPbeta and_GABPalpha. This mode of binding would be consistent with the observation that p16(INK4a) is sensitive to deleterious mutations found throughout this tumor suppressor protein; these mutations probably destabilize the three-dimensional structure.

Amino Acid Sequence↗
PubMed1998

New enzyme lineages by subdomain shuffling.

Protein functions have evolved in part via domain recombination events. Such events, for example, recombine structurally independent functional domains and shuffle targeting, regulatory, and/or catalytic functions. Domain recombination, however, can generate new functions, as implied by the observation of catalytic sites at interfaces of distinct folding domains. If useful to an evolving organism, such initially rudimentary functions would likely acquire greater efficiency and diversity, whereas the initially distinct folding domains would likely develop into single functional domains. This represents the probable evolution of the S1 serine protease family, whose two homologous beta-barrel subdomains assemble to form the binding sites and the catalytic machinery. Among S1 family members, the contact interface and catalytic residues are highly conserved whereas surrounding surfaces are highly variable. This observation suggests a new strategy to engineer viable proteins with novel properties, by swapping folding subdomains chosen from among protein family members. Such hybrid proteins would retain properties conserved throughout the family, including folding stability as single domain proteins, while providing new surfaces amenable to directed evolution or engineering of specific new properties. We show here that recombining the N-terminal subdomain from coagulation factor X with the C-terminal subdomain from trypsin creates a potent enzyme (fXYa) with novel properties, in particular a broad substrate specificity. As shown by the 2.15-A crystal structure, plasticity at the hydrophobic subdomain interface maintains activity, while surface loops are displaced compared with the parent subdomains. fXYa thus represents a new serine proteinase lineage with hybrid fX, trypsin, and novel properties.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Bis-substituted malonic acid hydroxamate derivatives as inhibitors of human neutrophil collagenase (MMP8).

Malonic acid hydroxamate derivatives bis-substituted at the methylene group were synthesized as potential nonpeptidic inhibitors of human neutrophil collagenase (MMP8). The presence of an aromatic residue both at the C2 malonic acid position and in the C-terminal tail for hydrophobic interactions with the surface-exposed S1 binding site and the S1' pocket of the enzyme, respectively, was found to be sufficient for submicromolar inhibition potencies. For optimal insertion of the aryl amide group into the hydrophobic S1' pocket, spacing of the C-terminal phenyl group by at least a 3C-chain was required. In view of these results the achiral indan-2, 2-dicarboxylic acid was used to mimic the 2-benzyl-2-methylmalonic acid residue, and its derivatization to the 3-phenylpropyl amide hydroxamate produced a potent, achiral, low-mass inhibitor of MMP8 (Ki = 0.3 microM), the binding mode of which was unambiguously determined by X-ray crystallographic analysis.

Binding Sites↗
PubMed1998

Diarylsulfonamides as selective, non-peptidic thrombin inhibitors.

Based on the structures of aminopyridine thrombin inhibitors (1), a series of aminoalkyl- and guanidinoalkyl-substituted diarylsulfonamides were prepared. The most potent derivative, N-[3-(4-guanidinobutoxy)-5-methyl-phenyl]-benzenesulfonamide (6c) had Ki = 0.18 microM for thrombin and did not inhibit trypsin, plasmin, or factor Xa. Comparison of the X-ray structures of the thrombin/1b and the thrombin/6c complexes revealed important aspects which govern the binding of such diarylsulfonamides to thrombin.

Antithrombins↗
PubMed1998

Structure of malonic acid-based inhibitors bound to human neutrophil collagenase. A new binding mode explains apparently anomalous data.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases, which have been implicated in various disease processes. Various classes of MMP inhibitors, including hydroxamic acids, phosphinic acids, and thiols, have been previously described. Most of these mimic peptides, and most likely bind analogous to the corresponding peptide substrates. Among the hydroxamic acids, malonic acid derivatives have been used as MMP inhibitors, although optimization of their inhibition potency was not successful. Here we report the design of malonic acid-based inhibitors using the X-ray structure of a collagenase/inhibitor complex, which revealed a nonsubstrate-like binding mode. The proposed beta-type turn-like conformation for the improved inhibitors was confirmed by X-ray crystallography. The observation of nonsubstrate-like binding confirms the original strategy for structure-based modeling of improved malonic acid inhibitors, and explains kinetic data that are inconsistent with substrate-like binding. Detailed interactions for the improved inhibitors seen in the crystal structure also suggest possibilities for further modifications in cycles of structure based drug design. Indeed, we have designed nonpeptidic inhibitors with approximately 500-fold improved inhibition based on these structures.

Collagenases↗
PubMed1997

Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential.

BACKGROUND: Staurosporine inhibits most protein kinases at low nanomolar concentrations. As most tyrosine kinases, along with many serine/threonine kinases, are either proto oncoproteins or are involved in oncogenic signaling, the development of protein kinase inhibitors is a primary goal of cancer research. Staurosporine and many of its derivatives have significant biological effects, and are being tested as anticancer drugs. To understand in atomic detail the mode of inhibition and the parameters of high-affinity binding of staurosporine to protein kinases, the molecule was cocrystallized with the catalytic subunit of cAMP-dependent protein kinase. RESULTS: The crystal structure of the protein kinase catalytic subunit with staurosporine bound to the adenosine pocket shows considerable induced-fit rearrangement of the enzyme and a unique open conformation. The inhibitor mimics several aspects of adenosine binding, including both polar and nonpolar interactions with enzyme residues, and induces conformational changes of neighboring enzyme residues. CONCLUSIONS: The results explain the high inhibitory potency of staurosporine, and also illustrate the flexibility of the protein kinase active site. The structure, therefore, is not only useful for the design of improved anticancer therapeutics and signaling drugs, but also provides a deeper understanding of the conformational flexibility of the protein kinase.

Amino Acid Sequence↗