Protein-inhibitor complexes, conformational

The overall structure of these metalloproteins is characterized by a deep active site cleft that divides the molecule into two domains, with the catalytically active zinc located at the bottom of the active site in the center of the molecule (Fig. 3). The structures of protein-inhibitor complexes [15,17,19-26] representing different families, indicate that the PI residue [37] is the principal recognition element for these enzymes (Fig. 5). Inhibitors bound to members of the matrixin family [17,19-23,25] adopt an extended conformation (Fig. 6), with the side chain at the P2 subsite directed away from the enz5mie on the opposite side of the inhibitor backbone to the PI specificity pocket. In addition to the zinc ligands, there are essential enzyme-inhibitor interactions between backbone atoms of the protein and complementary atoms of the inhibitor 1. 

The protease conformation of DegP is still elusive as crystallization of a substratelike inhibitor complex has failed and maintenance of a stably folded protein precludes long-term experimentation at elevated temperatures where it displays protease activity. We propose a profound rearrangement of the LA -L1-L2 loop triad into the canonical conformation of active serine proteases competent for substrate binding. This may be initiated by a collapse of the hydrophobic LA platforms and an enlargement of the hydrophobic contacts caused at high temperature. 

In the crystalline state these two forms are not isomorphous, and adding substrate to ConA crystals causes them to shatter.48 (This is a common observation and occurs even in hemoglobin crystals.) The differences between the conformers in the ConA part of the complex are not fully known, but there is considerable rearrangement of the protein. The rates of the reaction are fast, and so in solution the protein must be able to fluctuate readily between its different forms. Comparison with lysozyme (earlier in this article) shows that when lysozyme binds its inhibitors, a conformational change does occur, but it is not so gross that in the solid state shattering of crystals occurs. 

These results demonstrate that the side pocket of COX-1, which has been considered sterically inaccessible, can bind certain (5)-hydroxyethylamide derivatives of INDO. This study also offers insight into the selective binding of amide derivatives of INDO to COX-2. It seems that the amides associate with COX-1 and COX-2 but only bind tightly to COX-2. If all INDO-amides adopt a conformation in both enzymes analogous to that of the (i )-hydroxyethylamide 8, then this complex is only stable in COX-2. Because the orientation of Arg-120 is altered in the COX-1-compound 8 complex relative to other COX-1 inhibitor complexes, it may be that COX-2 can better accommodate this conformation than COX-1. It will be interesting to test this hypothesis and to identify the protein determinants responsible for the differential stability. 

The water-mediated interactions may also be of considerable importance. In the activated form the lid occupies a new position on the surface of the molecule, some 8 A away from the original location. This deep surface depression extends 10 A into the molecule and is filled in the native enzyme by 18 water molecules, half of which are direcdy hydrogen bonded to the polar protein groups. During the conformational change ail but three of these solvent molecules are expelled. In the lipase-inhibitor complex these three molecules become buried and mediate the polar contacts formed primarily by Asn-87. 

If thrombin and factor Xa, the major activated blood coagulation factors (Fig. 11.6), escape into healthy blood vessels, blood clots will develop and occlude capillaries throughout the body. Direct inhibition of these activated enzymes in the blood flow utilizes serine protease inhibitors, of which there are two common types a Kunitz inhibitor and a serpin. The former possess a Kunitz domain, a convex antiparallel (1-sheet that exactly fits into the concave active site of a serine protease, directly blocking it (lock and key mechanism). By contrast, serpins undergo complex interactions with other proteins to cause conformational changes that bait and block the catalytic action (Fig. 11.12 shows the bait). Table 11.3 fists the major coagulation inhibitors and cofactors, their targets and mechanisms of action. 

Many protein-ligand complexes are characterized by the presence of both polar and lipophilic interactions. The bound conformation of the ligand is determined by the relative importance of these contributions. An interesting example highlighting several important aspects was recenfly described by Lange and co-workers using the binding of non-peptidic inhibitors to the SH2 domain of src kinase [28]. The inhibitors are essentially tetrapeptide mimetics with tyrosine-phosphate or a... 

The complexities of developing a GTP-competitive therapeutic for Ras do not lie in its draggability. The active site of Ras is suitable for the isolation and optimization of such a compound. The first barrier is structural in nature A GTP/GDP-competitive inhibitor of Ras must necessarily promote the formation the inactive conformation of the protein or another conformation that is not recognizable to proteins which interact with Ras. A compound that stabilizes the formation of the active form of Ras would likely be highly oncogenic. The second barrier is thermodynamic the K. of Ras for GTP is 10 11 M and the concentration of GTP in... 

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