Non-conserved Amino Acid Residues Research Articles

Specificity in protein-protein recognition: conserved Im9 residues are the major determinants of stability in the colicin E9 DNase-Im9 complex.

The endonuclease group of E colicins are a family of bacterial toxins whose cytotoxic activity in a producing host is inactivated by a specific immunity protein. The DNase of colicin E9 can be bound and inhibited by both cognate and noncognate immunity proteins, the dissociation constants for which span a range of 12-orders of magnitude. DNase binding specificity of the immunity proteins is governed primarily by helix II, the sequence of which is variable in this family of proteins. Heteronuclear NMR experiments have identified helix III along with helix II as the likely DNase binding site, although other regions of Im9 also showed perturbations on binding the E9 DNase. In the present work, we have used the NMR experiments as a guide for alanine scanning mutagenesis of Im9. Our data show that helices II and III of Im9 are indeed the DNase binding site and in addition quantitate the relative binding energy associated with each helix. We find that the conserved residues of helix III make the largest relative contribution toward E9 DNase binding. In conjunction with previous studies, the data suggest that specificity in the colicin-immunity system is governed by a dual recognition mechanism in which highly stabilizing interactions emanating from the conserved regions of an immunity protein act as the binding site anchor and these are modulated by interactions from neighboring, nonconserved amino acid residues. This modulation is likely to take the form of both favorable and unfavorable interactions, the balance of which define the specificity of the protein-protein interaction. The generality of such a dual recognition mechanism in other systems is also discussed.

A Possible Role of the C-terminal Domain of the RecA Protein: A GATEWAY MODEL FOR DOUBLE-STRANDED DNA BINDING

According to the crystal structure, the RecA protein has a domain near the C terminus consisting of amino acid residues 270-328 (from the N terminus). Our model building pointed out the possibility that this domain is a part of "gateway" through which double-stranded DNA finds a path for direct contact with single-stranded DNA within a presynaptic RecA filament in the search for homology. To test this possible function of the domain, we made mutant RecA proteins by site-directed single (or double, in one case) replacement of 2 conserved basic amino acid residues and 5 among 9 nonconserved basic amino acid residues in the domain. Replacement of either of the 2 conserved amino acid residues caused deficiencies in repair of UV-damaged DNA, an in vivo function of RecA protein, whereas the replacement of most (except one) of the tested nonconserved ones gave little or no effect. Purified mutant RecA proteins showed no (or only slight) deficiencies in the formation of presynaptic filaments as assessed by various assays. However, presynaptic filaments of both proteins that had replacement of a conserved amino acid residue had significant defects in binding to and pairing with duplex DNA (secondary binding). These results are consistent with our model that the conserved amino acid residues in the C-terminal domain have a direct role in double-stranded DNA binding and that they constitute a part of a gateway for homologous recognition.

Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues. The cysteine residues of triosephosphate isomerase.

The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with triosephosphate isomerase. S-phenyl-p-toluenethiosulfonate (MePhSO2-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit triosephosphate isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit triosephosphate isomerase. The effect of MePhSO2-SPh on triosephosphate isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhSO2-SPh did not affect the activity of triosephosphate isomerase from S. cerevisiae and E. coli but it inhibited triosephosphate isomerase from chicken and S. pombe. From an analysis of the Cys content of the various triosephosphate isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSO2-SPh. Methyl metanethiosulfonate (MeSO2-SMe), which produces methyl disulfides, had no effect on triosephosphate isomerases that lack Cys217 (S. cerevisiae and E. coli). In triosephosphate isomerases that have Cys217, MeSO2-SMe inhibited by 40-50% the activity of that from S. pombe, 20-25% that from rabbit but had no effect on the chicken enzyme. In the three latter triosephosphate isomerases, MeSO2-SMe protected against the strong inhibiting action of MePhSO2-SPh. The latter observations suggest that MeSO2-SMe and MePhSO2-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit triosephosphate isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K(m) for glyceraldehyde 3-phosphate and a 65% decrease in Vmax. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cys14 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.

The role of the nonconserved residues at position 167 of class A beta-lactamases in susceptibility to mechanism-based inhibitors.

Differences in specificities between the class A beta-lactamases for both substrate and inhibitors are known. The role of the nonconserved amino acid residue at position 167 of the class A enzyme, which forms a cis bond with the catalytically essential Glu-166 residue, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors, was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace thr-167 with the corresponding Staphylococcus aureus PC1 residue Ile. Kinetic data obtained from the purified Thr-167-Ile B. cereus 569/H beta-lactamase was compared to that obtained from the wild-type B. cereus and S. aureus enzymes and indicated that the replacement had little effect on the Michaelis parameters for the hydrolysis of S- and A-type penicillins. However, the Thr-167-Ile enzymes became more S. aureus PC1-like in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone. A model for the role of this nonconserved residue at position 167 in the mechanism of inactivation by mechanism-based inhibitors is proposed.

A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functional analysis of nodE genes was constructed. By testing chimeric nodE genes constructed by the exchange of polymerase chain reaction (PCR)-generated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues). Mass spectrometric analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R. leguminosarum bv. viciae. The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C18:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively. Therefore, the main difference between the nodE-determined LCOs of biovar viciae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly developed spot application assay, we show that the C18:2- and C18:3-containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results, we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the polyunsaturated fatty acyl moieties of their LCOs, which is mediated by the host-specific central domain of the NodE protein.

A Comparison of the Enzymatic and Physicochemical Properties of Human Glutathione Transferase M4-4 and Three Other Human Mu Class Enzymes

The multigene family of cytosolic glutathione S-transferases (GSTs) consists of four classes (Alpha, Mu, Pi, and Theta), all involved in the detoxication of reactive electrophiles. The human Mu class GSTs consist of at least four expressed isozyme subunits, GST M1, GST M2, GST M3, and GST M4, which have 70-90% amino acid sequence identity. The gene and cDNA sequences for GST M4 have been determined recently (K. E. Comstock, K. J. Johnson, D. Rifenbery, and W. D. Henner, J. Biol. Chem. (1993) 268, 16958-16965). Cloning of GST M4 cDNA into an Escherichia coli expression system permitted the production of the corresponding protein. The enzyme was purified and shown to have a relatively low specific activity with the standard GST substrate 1-chloro-2,4-dinitrobenzene (1.4 ± 0.2 μmol min−1 mg−1 protein), but an activity equivalent to other Mu class enzymes with other tested substrates. The protein forms functional dimers composed of subunits with a Mr of approximately 26,400. A detailed comparison of the activity with various substrates and inhibitors was performed between GST M4-4 and other human Mu class GSTs, GST M1a-1a, GST M2-2, and GST M3-3, produced in bacterial expression systems. Despite the high level of amino acid sequence identity, the enzymatic properties of these enzymes were quite different. Comparisons with the crystallographic structure of a homologous rat GST, GST 3-3, indicate that a number of the nonconserved amino acid residues can be assigned to the putative active site of GST M4-4. This suggests that diversification in the evolution of these genes has occurred primarily in the substrate binding regions to cope with an increasing variety of foreign compounds.

Characterization of the antigenic and adhesive properties of FaeG, the major subunit of K88 fimbriae.

The two K88 serotypes, K88ab and K88ac, differ in terms of antigenic and adhesive properties. The structural determinants of the serotype-specific epitopes and the identify of the amino acid residues involved in fimbriae-receptor interaction were studied by the construction and analysis of K88 hybrid proteins in which various parts of the K88ab and K88ac fimbrial subunit FaeG were exchanged, and by in vitro mutagenesis of non-conserved amino acid residues. Using a set of monoclonal antibodies, several regions or amino acid residues involved in the formation of serotype-specific antigenic determinants were located. The haemagglutinating activity of the hybrid and mutant proteins revealed several amino acid residues involved in the formation of the receptor binding site. A clear correlation was found between the receptor binding site and the serotype-specific antigenic determinants.

The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

The C6 Zinc Finger and Adjacent Amino Acids Determine DNA-Binding Specificity and Affinity in the Yeast Activator Proteins LAC9 and PPR1

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Inhibition of the lytic activity of perforin (cytolysin) and of late complement components by proteoglycans

The complement components (C6, C7, C8 and C9) implicated in the lysis of target cells and the pore-forming, lytic protein from cytotoxic T-lymphocytes and NK-cells, perforin, contain an amino acid sequence which is highly homologous to a repeat unit identified in the LDL-receptor (Tschopp et al., 1986, Nature, 322, 831–834). The domain of the LDL-receptor, which is thought to interact with a positively charged segment of its ligands apoprotein B and E, is rich in cysteine residues and contains a cluster of negative charges. We show that the negatively charged molecules suramin and glycosaminoglycans, the positively charged peptides protamine and polylysine, all of which are known to abolish binding of LDL to its receptor (Goldstein et al, 1985, A. Rev. cell. Biol., 1, 1–39) inhibit the lytic activities of C6, C7, C8, C9 and perforin. Moreover, these negatively charged molecules are potent inhibitors of cytolytic T-lymphocyte-mediated lysis of target cells, suggesting a functionally crucial role for perforin in cell-mediated cytolysis. We propose that the negatively charged, cysteine-rich domain of these complement proteins and perforin interacts with an as yet unidentified positively charged segment of its ligand in a manner analogous to the LDL-LDL receptor interaction. Homologous cysteine-rich domains in functionally unrelated proteins may therefore be functionally conserved as ideal rigid interaction domains with the conserved cysteine residues as framework. Specificity of the domain for its ligand would be conferred by the non-conserved amino acid residues.