Crystal structure mutants

WA Lim, A Hodel, RT Sauer, FM Richards. The crystal structure of a mutant protein with altered but improved hydrophobic core packing. Proc Natl Acad Sci USA 91 423-427, 1994. PB Harbury, B Tidor, PS Kim. Repacking proteins cores with backbone freedom Structure prediction for coiled coils. Pi oc Natl Acad Sci USA 92 8408-8412, 1995. 

A second example is that of an Ala-to-Cys mutation, which causes the fonnation of a rare SH S hydrogen bond between the cysteine and a redox site sulfur and a 50 mV decrease in redox potential (and vice versa) in the bacterial ferredoxins [73]. Here, the side chain contribution of the cysteine is significant however, a backbone shift can also contribute depending on whether the nearby residues allow it to happen. Site-specific mutants have confirmed the redox potential shift [76,77] and the side chain conformation of cysteine but not the backbone shift in the case with crystal structures of both the native and mutant species [78] the latter can be attributed to the specific sequence of the ferre-doxin studied [73]. 

The elegant genetic studies by the group of Charles Yanofsky at Stanford University, conducted before the crystal structure was known, confirm this mechanism. The side chain of Ala 77, which is in the loop region of the helix-turn-helix motif, faces the cavity where tryptophan binds. When this side chain is replaced by the bulkier side chain of Val, the mutant repressor does not require tryptophan to be able to bind specifically to the operator DNA. The presence of a bulkier valine side chain at position 77 maintains the heads in an active conformation even in the absence of bound tryptophan. The crystal structure of this mutant repressor, in the absence of tryptophan, is basically the same as that of the wild-type repressor with tryptophan. This is an excellent example of how ligand-induced conformational changes can be mimicked by amino acid substitutions in the protein. 

Figure 17.14 Model of evolved mutant from cephalosphorinase shuffling. The sequence of the most active cephalosporinase mutant was modeled using the crystal structure of the class C cephalosporinase from Enterobacter cloacae. The mutant and wild-type proteins were 63% identical. This chimeric protein contained portions from three of the starting genes, including Enterobacter (blue), Klebsiella (yellow), and Citrobacter (green), as well as 33 point mutations (red). (Courtesy of A. Crameri.)...

The lipase (PAL) used in these studies is a hydrolase having the usual catalytic triad composed of aspartate, histidine, and serine [42] (Figure 2.6). Stereoselectivity is determined in the first step, which involves the formation of the oxyanion. Unfortunately, X-ray structural characterization of the (S)- and (J )-selective mutants are not available. However, consideration of the crystal structure of the WT lipase [42] is in itself illuminating. Surprisingly, it turned out that many of the mutants have amino acid exchanges remote from the active site [8,22,40]. 

The ANEH-mutant displaying enhanced enantioselectivity ( =10.8) was sequenced and shown to be characterized by three mutations, A217V near the active site and K332E and A390E both at remote positions [58]. The X-ray crystal structure of the WT ANEH had been analyzed earlier [61], revealing a dimer comprising identical... 

While wild-type PAMO was unable to convert 2-phenylcyclohexanone efficiently, all deletion mutants readily accepted this ketone as substrate. All mutants also displayed a similar thermostability when compared with the parent enzyme. The most active mutant (deletion of S441 and A442) was used for examining its enantioselective properties. It was found that the mutant preferably formed the (/ )-enantiomer of the corresponding lactone E = 100). While CHMO also shows a similar enantioselective behavior, this PAMO deletion mutant is a better candidate for future applications due to its superior stability. This clearly demonstrates that PAMO can be used as parent enzyme to design thermostable BVMO variants. It also illustrates that the available crystal structure of PAMO will be of great help for BVMO redesign efforts. ... 

Both enzymes belong to the family of a,p-hydrolases." The active site of MeHNL is located inside the protein and connected to the outside through a small channel, which is covered by the bulky amino acid tryptophane 128." It was possible to obtain the crystal structure of the complex with the natural substrate acetone cyanohydrin with the mutant SerSOAla of MeHNL. This complex allowed the determination of the mode of substrate binding in the active site." A summary of 3D structures of known HNLs was published recently." " ... 

TKase is a homodimeric protein with a subunit of about 70kDa. The X-ray structures of TKase of E. colif S. cerevisiaeX Leishmania mexicana and mize have been solved. In addition, the crystal structures of a number of site-directed mutants have been determined. Schneider and co-workers have reported a series of studies in which they have mutated important residues of active site of TKase to elucidate the reaction mechanism and explain the origin of the stereospecificity of the C—C bond-forming process (Table The conserved... 

G. F. Maley, P. Van Roey 2001, (Crystal structure of a deletion mutant of human thymidylate synthase Delta(7-29) and its ternary complex with Tomudex and dUMP), Protein Sci. 10(5), 988. 

This approach is not restricted to bacterial or viral cells. Mammalian cells under highly proliferating conditions can be cultured at increasing exposure to a compound in attempts to create resistant mutants. Alternatively, one can sometimes use a structural biology approach to predict amino acid changes that would abrogate inhibitor affinity from study of enzyme-inhibitor complex crystal structures. If the recombinant mutant enzyme displays the diminished inhibitor potency expected, one can then devise ways of expressing the mutant enzyme in a cell type of interest and look to see if the cellular phenotype is likewise abolished by the mutation. 

D. Erion, S- J- Pilkis, M. R. El-Maghrabi, and W. N. Lipscomb, The allosteric site of human liver fructose 1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure, J. Biol. Chem. 269 27732 (1994). 

Janssen, P. L Boyer, P. Clark, R. H. Smith, M. B. Kroeger Smith, C. J. Michejda, S.. Hughes, and E. Arnold, Crystal structures of 8-C1 and 9-C1 TIBO complexed with wild-type HIV-1 RT and 8-C1 TIBO with the Tyrl81Cys HIV-1 RT drug-resistant mutant, J. Mol. Biol. 264 1085 (1996). 

L. Hong, A. Treharne, J. A. Hartsuck, S. Foundling, J. Tang, Crystal Structures of Complexes of a Peptidic Inhibitor with Wild Type and Two Mutants HIV-1 Proteases , Biochemistry 1996, 35, 10627-10633. 

Fields, B.A., F.A. Goldbaum, W. Dall Acqua, E.L. Malchiodi, A. Cauerhff, F.P. Schwarz, X. Ysem, R.J. Poljak, and R.A. Mariuzza. 1996. Hydrogen bonding and solvent structure in an antigen-antibody interface. Crystal structures and thermodynamic characterization of three Fv mutants complexed with lysozyme. Biochemistry 35 15494-15503. 

One of the important consequences of studying catalysis by mutant enzymes in comparison with wild-type enzymes is the possibility of identifying residues involved in catalysis that are not apparent from crystal structure determinations. This has been usefully applied (Fersht et al., 1988) to the tyrosine activation step in tyrosine tRNA synthetase (47) and (49). The residues Lys-82, Arg-86, Lys-230 and Lys-233 were replaced by alanine. Each mutation was studied in turn, and comparison with the wild-type enzyme revealed that each mutant was substantially less effective in catalysing formation of tyrosyl adenylate. Kinetic studies showed that these residues interact with the transition state for formation of tyrosyl adenylate and pyrophosphate from tyrosine and ATP and have relatively minor effects on the binding of tyrosine and tyrosyl adenylate. However, the crystal structures of the tyrosine-enzyme complex (Brick and Blow, 1987) and tyrosyl adenylate complex (Rubin and Blow, 1981) show that the residues Lys-82 and Arg-86 are on one side of the substrate-binding site and Lys-230 and Lys-233 are on the opposite side. It would be concluded from the crystal structures that not all four residues could be simultaneously involved in the catalytic process. Movement of one pair of residues close to the substrate moves the other pair of residues away. It is therefore concluded from the kinetic effects observed for the mutants that, in the wild-type enzyme, formation of the transition state for the reaction involves a conformational change to a structure which differs from the enzyme structure in the complex with tyrosine or tyrosine adenylate. The induced fit to the transition-state structure must allow interaction with all four residues simultaneously. 

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