Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, JBiol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution.The structure of the Co2+-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobaltrelative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd2+-and Mn2+-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 1 4 4 . Some of these sidechain movements are similar to adjustments that have been observed previously in association with the "hingebending" motion that is presumed to occur during catalysis by the zinc endoproteases.In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect. (Fig. 1) showed the zinc liganded tetrahedrally by His 142, His 146, Glu 166, and a water molecule, hereafter referred to as Wat 231 (Fig. 2). Further refinement has suggested that, in addition to Wat 23 1, there is also a dipeptide, presumably a product of selfproteolysis, bound to the active site (Holland et al., 1992 (1974, 1976) have shown that removal of zinc from thermolysin yields an inactive apoenzyme and that varying levels of esterase and peptidase activity can be restored upon substitution of the native zinc atom with transition metals. In particular, Zn2+, Co2+, and Mn2+, when added in stoichiometric amounts, restored 100,200, and 10% of the activity of the native enzyme toward furylacyloylglycyl-L-leucyl amide (FAGLA). Furthermore, in concentrations up to 1 mM, these metals retained or restored catalytic activity to some extent. Fe2+ in high molar excess restored about 60% of native activity. Zn2+ in excess of the amount required for catalytic activity actually inhibited the enzyme. The transition metals Mg2+, Cr2+, Ni2+, Cu2+, Mo2+, Pb2+, Hg2+, Cd2+, Nd2+...

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function.The small heat shock proteins (sHSPs) 1 and the structurally related vertebrate eye lens ␣-crystallins are a family of virtually ubiquitous stress proteins (1, 2). Their role in cellular stress extends from protection against high temperature and oxidative stress to a potentially important function in a variety of protein-folding diseases and aging (3, 4). The sHSPs are defined by a conserved C-terminal domain of ϳ90 amino acids (the ␣-crystallin domain), which is flanked by a variable length N-terminal arm and a more conserved C-terminal extension (1, 5). These small proteins (16 -42-kDa monomers) assemble into oligomeric structures of 9 to Ͼ32 subunits depending on the sHSP (6, 7). sHSPs are very efficient at binding denatured proteins, and current models propose that they function as ATP-independent molecular chaperones to prevent irreversible protein aggregation and insolubilization (2). sHSP-bound proteins can be reactivated by the ATP-dependent chaperone activity of DnaK/Hsp70, with the help of ClpB or GroEL in some instances (8 -10). Thus, sHSPs are proposed to be a critical component of the cellular chaperone network that becomes particularly important under conditions of severe stress involving protein aggregation.Although the ability of sHSPs to interact with denatured model substrates in vitro has been studied extensively, the characteristics of cellular sHSP substrates remain poorly defined. Understanding the mechanism of sHSP chaperone action, as well as determining how these proteins may act to protect cells during stress, requires identification of proteins that interact with sHSPs in vivo, either as partners or subst...

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The periplasmic protein CusF, as a part of the CusCFBA efflux complex, plays a role in resistance to elevated levels of copper and silver in Escherichia coli. Although homologues have been identified in other Gram-negative bacteria, the substrate of CusF and its precise role in metal resistance have not been described. Here, isothermal titration calorimetry (ITC) was used to demonstrate that CusF binds with high affinity to both Cu(I) and Ag(I) but not Cu(II). The affinity of CusF for Ag(I) was higher than that for Cu(I), which could reflect more efficient detoxification of Ag(I) given the lack of a cellular need for Ag(I). The chemical shifts in the nuclear magnetic resonance (NMR) spectra of CusF-Ag(I) as compared to apo-CusF show that the region of CusF most affected by Ag(I) binding encompasses three absolutely conserved residues: H36, M47, and M49. This suggests that these residues may play a role in Ag(I) coordination. The NMR spectra of CusF in the presence of Cu(II) do not indicate specific binding, which is in agreement with the ITC data. We conclude that Cu(I) and Ag(I) are the likely physiological substrates.