Protein-DNA interaction. No code for recognition.
Explore the source record for details and available documents.
DNA↗
Biomedical subjects
B W Matthews
Publications and source records attributed to B W Matthews.
Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3.
Replacing the isoleucine at amino-acid position three of bacteriophage T4 lysozyme causes changes in the thermodynamic stability of the protein that are directly related to the hydrophobicity of the substituted residue. Structural analysis confirms that the hydrophobic stabilization is proportional to the reduction of the surface area accessible to solvent on folding.
Amino Acids↗
The binding of L-valyl-L-tryptophan to crystalline thermolysin illustrates the mode of interaction of a product of peptide hydrolysis.
Crystallographic analysis of the binding of mercaptoacetyl-L-valyl-L-tryptophan to thermolysin suggests that this inhibitor is hydrolyzed by the crystalline enzyme. The apparent product of hydrolysis, L-valyl-L-tryptophan (Val-Trp), occupies the S1'-S2' subsites of the active site, not the S1-S1' subsites as observed previously for the dipeptide L-alanyl-L-phenylalanine (Ala-Phe). The difference in binding of Val-Trp and Ala-Phe is consistent with the specificity requirements and preferences of thermolysin. The binding of Val-Trp illustrates the mode of interaction of one of the products of peptide hydrolysis. High resolution crystallographic refinement indicates that the valyl amino group makes three hydrogen bonds to the enzyme and to solvent and, in addition, is 2.8 A from the carboxylate of Glu-143. This is the first instance in which a direct interaction has been observed between Glu-143 and the scissile nitrogen. As such, the study directly supports the mechanism of action for thermolysin proposed by Hangauer et al. (Hangauer, D. G., Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5730-5741) and, by analogy, indirectly supports the similar mechanism proposed for carboxypeptidase A (Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5724-5729).
Binding Sites↗
Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability.
To investigate the relation between protein stability and the predicted stabilities of individual secondary structural elements, residue Pro86 in an alpha-helix in phage T4 lysozyme was replaced by ten different amino acids. The x-ray crystal structures of seven of the mutant lysozymes were determined at high resolution. In each case, replacement of the proline resulted in the formation of an extended alpha-helix. This involves a large conformational change in residues 81 to 83 and smaller shifts that extend 20 angstroms across the protein surface. Unexpectedly, all ten amino acid substitutions marginally reduce protein thermostability. This insensitivity of stability to the amino acid at position 86 is not simply explained by statistical and thermodynamic criteria for helical propensity. The observed conformational changes illustrate a general mechanism by which proteins can tolerate mutations.
Enzyme Stability↗
Slow- and fast-binding inhibitors of thermolysin display different modes of binding: crystallographic analysis of extended phosphonamidate transition-state analogues.
The modes of binding to thermolysin of two phosphonamidate peptide inhibitors, carbobenzoxy-GlyP-L-Leu-L-Leu (ZGPLL) and carbobenzoxy-L-PheP-L-Leu-L-Ala (ZFPLA), have been determined by X-ray crystallography and refined at high resolution to crystallographic R-values of 17.7% and 17.0%, respectively. (GlyP is used to indicate that the trigonal carbon of the peptide linkage is replaced by the tetrahedral phosphorus of a phosphonamidate group.). These inhibitors were designed to be structural analogues of the presumed catalytic transition state and are potent inhibitors of thermolysin (ZGPLL, Ki = 9.1 nM; ZFPLA, Ki = 0.068 nM) [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. ZFPLA binds to thermolysin in the manner expected for the transition state and, for the first time, provides direct support for the presumed mode of binding of extended substrates in the S2 subsite. The mode of binding of ZFPLA displays all the interactions that are presumed to stabilize the transition state and supports the postulated mechanism of catalysis [Hangauer, D. G., Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry 23, 5730-5741]. The two oxygens of the phosphonamidate moiety are liganded to the zinc to give overall pentacoordination of the metal. For the second inhibitor the situation is different. Although both ZFPLA and ZGPLL have similar modes of binding in the S1' and S2' subsites, the configurations of the carbobenzoxy-Phe and carbobenzoxy-Gly moieties are different. For ZFPLA the carbonyl group of the carbobenzoxy group is hydrogen bonded directly to the enzyme, whereas in ZGPLL the carbonyl group is rotated 117 degrees, and there is a water molecule interposed between the inhibitor and the enzyme. For ZGPLL only one of the phosphonamidate oxygens is liganded to the zinc. Correlated with the change in inhibitor-zinc ligation from monodentate in ZGPLL to bidentate in ZFPLA there is an increase in the phosphorus-nitrogen bond length of about 0.25 A, strongly suggesting that the phosphonamide nitrogen in ZFPLA is cationic, analogous to the doubly protonated nitrogen of the transition state. The observation that the nitrogen of ZFPLA appears to donate two hydrogen bonds to the protein also indicates that it is cationic. The different configurations adopted by the respective inhibitors are correlated with large differences in their kinetics of binding [Bartlett, P. A., & Marlowe, C. K. (1987) Biochemistry (following paper in this issue)]. These differences in kinetics are not associated with any significant conformational change on the part of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Binding Sites↗
Structural analysis of the temperature-sensitive mutant of bacteriophage T4 lysozyme, glycine 156----aspartic acid.
The structure of the mutant of bacteriophage T4 lysozyme in which Gly-156 is replaced by aspartic acid is described. The lysozyme was isolated by screening for temperature-sensitive mutants and has a melting temperature at pH 6.5 that is 6.1 degrees C lower than wild type. The mutant structure is destabilized, in part, because Gly-156 has conformational angles (phi, psi) that are not optimal for a residue with a beta-carbon. High resolution crystallographic refinement of the mutant structure (R = 17.7% at 1.7 A resolution) shows that the Gly----Asp substitution does not significantly alter the configurational angles (phi, psi) but forces the backbone to move, as a whole, approximately 0.6 A away from its position in wild-type lysozyme. This induced strain weakens a hydrogen bond network that exists in the wild-type structure and also contributes to the reduced stability of the mutant lysozyme. The introduction of an acidic side chain reduces the overall charge on the molecule and thereby tends to increase the stability of the mutant structure relative to wild type. However, at neutral pH this generalized electrostatic stabilization is offset by specific electrostatic repulsion between Asp-156 and Asp-92. The activity of the mutant lysozyme is approximately 50% that of wild-type lysozyme. This reduction in activity might be due to introduction of a negative charge and/or perturbation of the surface of the molecule in the region that is assumed to interact with peptidoglycan substrates.
Aspartic Acid↗
Genetic and structural analysis of the protein stability problem.
Explore the source record for details and available documents.
Calorimetry↗
Structural studies of mutants of the lysozyme of bacteriophage T4. The temperature-sensitive mutant protein Thr157----Ile.
To understand the roles of individual amino acids in the folding and stability of globular proteins, a systematic structural analysis of mutants of the lysozyme of bacteriophage T4 has been undertaken. The isolation, characterization, crystallographic refinement and structural analysis of a temperature-sensitive lysozyme in which threonine 157 is replaced by isoleucine is reported here. This mutation reduces the temperature of the midpoint of the reversible thermal denaturation transition by 11 deg.C at pH 2.0. Electron density maps showing differences between the wild-type and mutant X-ray crystal structures have obvious features corresponding to the substitution of threonine 157 by isoleucine. There is little difference electron density in the remainder of the molecule, indicating that the structural changes are localized to the site of the mutation. High-resolution crystallographic refinement of the mutant lysozyme structure confirms that it is very similar to wild-type lysozyme. The largest conformational differences are in the gamma-carbon of residue 157 and in the side-chain of Asp159, which shift 1.0 A and 1.1 A, respectively. In the wild-type enzyme, the gamma-hydroxyl group of Thr157 participates in a network of hydrogen bonds. Substitution of Thr157 with an isoleucine disrupts this set of hydrogen bonds. A water molecule bound in the vicinity of Thr155 partially restores the hydrogen bond network in the mutant structure, but the buried main-chain amide of Asp159 is not near a hydrogen bond acceptor. This unsatisfied hydrogen-bonding potential is the most obvious reason for the reduction in stability of the temperature-sensitive mutant protein.
Crystallography↗
Kinetic studies on Cro repressor-operator DNA interaction.
The six operators of phage lambda and their consensus sequence were synthesized as 21 base-pair DNAs and their interactions with Cro repressor were studied using a filter binding assay. The measured equilibrium dissociation constants suggest that Cro has the highest affinity to the consensus operator (KD = 1.2 X 10(-12) M) and then the OR3 operator (KD = 2.0 X 10(-12) M), after that the affinity becomes lower in the following order: OR1, OL1, OL2, OL3, OR2. The competition experiments show that Cro forms the most stable complex with the consensus operator (t1/2 = 150 min), which is followed by the complex with OR3 (t1/2 = 70 min), OR1, OL1, OL2, OL3 and OR2. The association rate constants (ka) were also measured. They are approximately the same (2 X 10(8) to 4 X 10(8) m-1 s-1) for the consensus, OR3, OR2 and OR1 operators. These experiments have thus shown that the sequence difference in the operator affects the dissociation (KD and kd) but not the association (ka) process. The operators' binding strengths relative to OR1 are 14 (for consensus operator), 7.6 (OR3), 0.73 (OL1), 0.42 (OL2), 0.16 (OL3) and 0.1 (OR2). Seven different lengths of OR-containing DNA fragments were prepared. Measurement of kinetic parameters shows that the affinity of Cro to operator DNA (measured by KD) is essentially constant and independent of the DNA length, while the association and dissociation rate constants increase as the DNA length increases. This is consistent with the idea that Cro locates and leaves its operator via a two-step mechanism. It appears that Cro binds first at an arbitrary site on DNA, then is transferred to its operator site by a facilitated mechanism. The process is reversed when Cro dissociates from the operator. Most of our data fit to the theoretical expression formulated by Berg, Winter & von Hippel for the sliding mechanism. We conclude that Cro slides along the DNA to locate and leave the operator.
Bacteriophage lambda↗
Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein.
Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.
2-Aminopurine↗
Structures of two thermolysin-inhibitor complexes that differ by a single hydrogen bond.
The mode of binding to thermolysin of the ester analog Cbz-GlyP-(O)-Leu-Leu has been determined by x-ray crystallography and shown to be virtually identical (maximum difference 0.2 angstrom) with the corresponding peptide analog Cbz-GlyP-(NH)-Leu-Leu. The two inhibitors provide a matched pair of enzyme-inhibitor complexes that differ by 4.1 kilocalories per mole in intrinsic binding energy but are essentially identical except for the presence or absence of a specific hydrogen bond.
Amides↗
Structure of bacteriophage T4 lysozyme refined at 1.7 A resolution.
The structure of the lysozyme from bacteriophage T4 has been refined at 1.7 A resolution to a crystallographic residual of 19.3%. The final model has bond lengths and bond angles that differ from "ideal" values by 0.019 A and 2.7 degrees, respectively. The crystals are grown from electron-dense phosphate solutions and the use of an appropriate solvent continuum substantially improved the agreement between the observed and calculated structure factors at low resolution. Apart from changes in the conformations of some side-chains, the refinement confirms the structure of the molecule as initially derived from a 2.4 A resolution electron density map. There are 118 well-ordered solvent molecules that are associated with the T4 lysozyme molecule in the crystal. Four of these are more-or-less buried. There is a clustering of water molecules within the active site cleft but, other than this, the solvent molecules are dispersed around the surface of the molecule and do not aggregate into ice-like structures or pentagonal or hexagonal clusters. The apparent motion of T4 lysozyme in the crystal can be interpreted in terms of significant interdomain motion corresponding to an opening and closing of the active site cleft. For the amino-terminal domain the motion can be described equally well (correlation coefficients approx. 0.87) as quasi-rigid-body motion either about a point or about an axis of rotation. The motion in the crystals of the carboxy-terminal domain is best described as rotation about an axis (correlation coefficient 0.80) although in this case the apparent motion seems to be influenced in part by crystal contacts and may be of questionable relevance to dynamics in solution.
Amino Acid Sequence↗
Structure and thermal stability of phage T4 lysozyme.
Explore the source record for details and available documents.
Enzyme Stability↗
Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding.
It is proposed that the stability of a protein can be increased by selected amino acid substitutions that decrease the configurational entropy of unfolding. Two such substitutions, one of the form Xaa----Pro and the other of the form Gly----Xaa, were constructed in bacteriophage T4 lysozyme at sites consistent with the known three-dimensional structure. Both substitutions stabilize the protein toward reversible and irreversible thermal denaturation at physiological pH. The substitutions have no effect on enzymatic activity. High-resolution crystallographic analysis of the proline-containing mutant protein (Ala-82----Pro) shows that its three-dimensional structure is essentially identical with the wild-type enzyme. The overall structure of the other mutant enzyme (Gly-77----Ala) is also very similar to wild-type lysozyme, although there are localized conformational adjustments in the vicinity of the altered amino acid. The combination of a number of such amino acid replacements, each of which is expected to contribute approximately 1 kcal/mol (1 cal = 4.184 J) to the free energy of folding, may provide a general strategy for substantial improvement in the stability of a protein.
Calorimetry↗
Different interactions used by Cro repressor in specific and nonspecific DNA binding.
The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation. However, the terminal amino group and lysines 8, 18, and 39 are not protected. Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region. The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf, D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758; Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews, B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove. Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding. Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic. On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well. Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.
Amino Acid Sequence↗
Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin.
The mode of binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide (P-Leu-NH2) has been determined crystallographically and refined at high resolution (R = 17.9% to 0.16-nm resolution). The mode of binding of the naturally occurring thermolysin inhibitor phosphoramidon reported previously [Weaver, L. H., Kester, W. R. and Matthews, B. W. (1977) J. Mol. Biol. 114, 119-132] has also been confirmed by crystallographic refinement (R = 17.4% to 0.23-nm resolution). Phosphoramidon binds to the enzyme with a single oxygen of the phosphoramidate moiety as a zinc ligand. Together with three ligands to the metal from the protein the resultant complex has approximately tetrahedral geometry. However, in the case of P-Leu-NH2, two of the phosphoramidate oxygens interact with the zinc to form a complex that tends towards pentacoordinate. In this respect, P-Leu-NH2 appears to be a better transition-state analog than is phosphoramidon. In addition, the phosphorus-nitrogen bond length in P-Leu-NH2 is 0.18 nm, suggesting that the nitrogen is protonated whereas the same bond in phosphoramidon is much shorter (0.15 nm) suggesting that the nitrogen does not carry a charge. In phosphoramidon the distance from the phosphoramide nitrogen to Glu-143 is 0.39 nm whereas in P-Leu-NH2 this distance decreases to 0.34 nm. Taken together, these observations provide additional evidence in support of the participation of pentacoordinate intermediates in the mechanism of action of thermolysin [Holmes, M. A. and Matthews, B. W. (1981) Biochemistry 20, 6912-6920] and the role of Glu-143 in first promoting the attack of a water molecule on the carbonyl carbon of the scissile bond and subsequently acting as a 'proton shuttle' to transfer the proton to the leaving nitrogen [Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5724-5729; Hangauer, D. G., Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5730-5741].
Crystallography↗
Structure and X-ray amino acid sequence of a bacteriochlorophyll A protein from Prosthecochloris aestuarii refined at 1.9 A resolution.
The structure of the water-soluble bacteriochlorophyll a protein (Bchl protein) from the green photosynthetic bacterium Prosthecochloris aestuarii has been refined at 1.9 A resolution to a crystallographic residual of 18.9%. The refinement was carried out without knowledge of the amino acid sequence and has led to an "X-ray sequence". The structure consists of seven Bchl molecules enclosed within a protein "bag" and the refinement supports the general conformation of the molecule described previously. The refinement also supports the previous suggestion that the ligands to the seven Bchl magnesiums are, respectively, five histidines, a carbonyl oxygen from the polypeptide backbone of the protein, and a bound water molecule. The conformations of the seven Bchl head-groups are described in detail. In two cases the magnesium atoms are approximately 0.48 A "below" the plane of the conjugated macrocycle while in the other five cases the atoms are, on average, 0.48 A "above" the plane. The acetyl ring substituents are more-or-less coplanar with the dihydrophorbin macrocycle, consistent with a previous resonance Raman study. The conjugated atoms in each of the seven macrocycles have significant departures from strict planarity. These deviations are similar for Bchls 1, 2 and 3 (class I) and are also similar for Bchls 4, 5, 6 and 7 (class II). Ethylchlorophillide also belongs to class II. The out-of-plane deformations for the class I and class II bacteriochlorophylls appear to correspond to two distinct modes of bending or curvature of the dihydrophorbin macrocycle.
Amino Acid Sequence↗
Crystallization of a complex of cro repressor with a 17 base-pair operator.
Crystals of the lambda cro repressor complexed to a 17 base-pair synthetic binding site related to the OR3 operator have been obtained. The complex crystallizes in the hexagonal space group P6(2) (or P6(4)) with unit cell dimensions a = b = 154.8 A, c = 85.6 A. Preliminary photography reveals that the crystals are stable to X-rays and display measurable reflections to a resolution of about 3.7 A. The diffraction patterns suggest that the cro-DNA complexes are arranged in an open hexagonal network with the DNA fragments stacked end-to-end. The DNA is in the B-form but appears to be bent or curved into an approximate superhelix.
Bacteriophage lambda↗