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T A Steitz

Publications and source records attributed to T A Steitz.

At least 145 records · Page 8Linked to original sources
PubMed1982

Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP.

The amino acid sequence of the Escherichia coli catabolite gene activator protein has been fit into a 2.9-A resolution electron density map. Each subunit of the dimer consists of two structurally distinct domains. The larger NH2-terminal domain is seen to bind cyclic AMP and forms all of the contacts between the subunits. The cyclic AMP is completely buried between the interior of the "beta roll" structure of the large domain and a long alpha helix; it makes important hydrogen-bonding interactions with residues from both subunits. The guanidinium group of a buried Arg makes an internal salt link with the phosphate of cyclic AMP. The 6-amino group of adenine interacts simultaneously with both subunits. This interaction with both subunits and the fact that cyclic GMP and cyclic IMP do not activate catabolite gene activator protein suggest that the binding of cyclic AMP may alter the relative orientation of the two subunits, which in turn would change the structure of a DNA binding site that is presumed to span the two smaller domains. The distribution and nature of side chains in the small domain do not rule out the possibility that catabolite gene activator protein binds to left-handed B-DNA.

Allosteric Regulation↗
PubMed1982

The 6-hydroxymethyl group of a hexose is essential for the substrate-induced closure of the cleft in hexokinase.

Yeast hexokinase B (ATP:-hexose 6-phosphotransferase, EC 2.7.1.1) was crystallized in the presence of D-xylose and ADP, and its structure was determined at 7 A resolution. The enzyme is in the 'open' conformation which is characteristic of the enzyme crystallized in the absence of glucose, rather than in the 'closed' conformation that is observed with the glucose complex. That is, the binding of xylose into the large cleft that separates the molecule into two lobes does not cause the cleft to close. We conclude, then, that the glucose 6-hydroxymethyl group (which binds to an aspartic acid and a serine) is essential for the hexose-induced conformational change.

Binding Sites↗
PubMed1982

Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins.

It is shown that there is a structural similarity between the presumed DNA-binding regions of the Escherichia coli catabolite gene activator protein ("CAP") and the cro repressor protein ("cro") from bacteriophage lambda. The correspondence between the two proteins is particularly striking for a structural unit consisting of two consecutive alpha-helices. The 24 alpha-carbon atoms that constitute the two-helical structural units in the two proteins can be superimposed with a root-mean-square disagreement of 1.1 A. It is shown that this agreement is very unlikely to be due to a chance correspondence. For both CAP activator and cro repressor proteins it is the second alpha-helix of the two-helical unit that has been proposed to bind within the major groove of left-handed or right-handed B DNA, respectively [McKay, D. B. & Steitz, T. A. (1981) Nature (London) 290, 744-749; Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B. W. (1981) Nature (London) 290, 754-758]. The structural correspondence between CAP and cro seen here, together with other recent evidence of sequence homologies between cro, CAP, and other proteins that bind double-stranded DNA, suggests that the two-helical unit is likely to be a common feature of many DNA-binding proteins. The results also suggest that some principles of specific protein-double-stranded DNA interaction may be general and include recognition via alpha-helices fitting into the major groove of the DNA.

Bacteriophage lambda↗
PubMed1982

The cAMP-binding domains of the regulatory subunit of cAMP-dependent protein kinase and the catabolite gene activator protein are homologous.

Comparison of the recently determined amino acid sequences of the regulatory subunit of cAMP-dependent protein kinase (RII) from bovine cardiac muscle and the Escherichia coli catabolite gene activator protein (CAP) shows significant homology. This homology extends over most of the amino-terminal domain in CAP and is particularly good for the region of the beta-roll structure. The RII sequence contains two adjacent and internally homologous regions, both of which have high resemblance to the cAMP-binding domain in CAP. This suggests that the protein kinase regulatory subunit contains two cAMP-binding domains in the carboxyl-terminal region, each having a beta-roll structure similar to that in CAP. The cAMP molecule is expected to bind to the RII within a pocket formed by residues from the beta-roll, as is the case with CAP. One cAMP molecule would interact with residues from about 163 to 220, and the other cAMP would interact with amino acids in the stretch 285-350 of the RII protein kinase sequence. As the carboxyl-terminal domain of CAP shows homologies to the DNA-binding domains of other regulatory proteins, the protein appears to be of modular construction: a DNA-binding domain joined to a cAMP-binding domain.

Amino Acid Sequence↗
PubMed1981

Structural dynamics of yeast hexokinase during catalysis.

The binding of the substrate glucose to yeast hexokinase results in a substantial enzyme conformational change that is essential for catalysis and may be important for the enzyme's specificity, as well as the control of its activity. From high-resolution crystal structures of the monomeric enzyme crystallized both in the presence and in the absence of glucose, we find that glucose binds into the deep cleft that separates the molecule into two lobes and causes these two lobes to move together and close off the cleft. The structure of the hexokinase crystallized in the presence of xylose and ADP is being determined at low resolution. In this crystal form, the enzyme was thought to be in the conformation of the ternary complex. However, a low-resolution structure of this crystal form shows clearly that the enzyme is in the 'open' form and is not a ternary complex. Crystals of the A isozyme with glucose and ADP may be. Further, chemically sequenced tryptic peptides are being incorporated into the model obtained by crystallographic refinement at 2.1 A resolution. Completion of the sequence and the structure of the ternary complex should allow a detailed description of the enzymatic mechanism of this kinase and the role of substrate-induced conformational changes in catalysis and control.

Adenosine Triphosphate↗
PubMed1981

Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA.

The 2.9 A resolution crystal structure of Escherichia coli catabolite gene activator protein (CAP) complexed with cyclic AMP reveals two distinct structural domains separated by a cleft. The smaller carboxy-terminal domain is presumed to bind DNA while the amino-terminal domain is seen to bind cyclic AMP. Model building studies suggest that CAP binds to left-handed B-type DNA, contracting its major groove via two alpha-helices. It is possible that the CAP conversion of right- to left-handed DNA in a closed supercoil, is what activates transcription by RNA polymerase.

Carrier Proteins↗
PubMed1981

The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis.

We propose that the initial event in the secretion of proteins across membranes and their insertion into membranes is the spontaneous penetration of the hydrophobic portion of the bilayer by a helical hairpin. Energetic considerations of polypeptide structures in a nonpolar, lipid environment compared with an aqueous environment suggest that only alpha and 3(10) helices will be observed in the hydrophobic interior of membranes. Insertion of a polypeptide is accomplished by a hairpin structure composed of two helices, which will partition into membranes if the free energy arising from burying hydrophobic helical surfaces exceeds the free energy "cost" of burying potentially charged and hydrogen-bonding groups. We suggest, for example, that the hydrophobic leader peptide found in secreted proteins and in many membrane proteins forms one of these helices and is oriented in the membrane with its N terminus inside. In secreted proteins, the leader functions by pulling polar portions of a protein into the membrane as the second helix of the hairpin. The occurrence of all categories of membrane proteins can be rationalized by the hydrophobic or hydrophilic character of the two helices of the inserted hairpin and, for some integral membrane proteins, by events in which a single terminal helix is inserted. We propose that, because of the distribution of polar and nonpolar sequences in the polypeptide sequence, secretion and the insertion of membrane proteins are spontaneous processes that do not require the participation of additional specific membrane receptors or transport proteins.

Biological Transport↗
PubMed1979

Substrate binding closes the cleft between the domains of yeast phosphoglycerate kinase.

Using small angle x-ray scattering from solutions of yeast phosphoglycerate kinase, we have measured the radius of gyration of the enzyme both in the presence and in the abscence of ligands. We find that the radius of gyration decreases by 1.09 +/- 0.34 A upon binding both substrates MgATP and 3-phosphoglycerate to form the ternary complex. Smaller decreases, at the limit of the precision of the measurement, were found for the separate binding of MgATP (0.30 +/- 0.50 A). Using computer modeling, it has been estimated that a substrate-induced cleft closure in phosphoglycerate kinase resulting from one lobe rotating 8-14 degrees relative to the other lobe lobe is consistent with this observed change in radius of gyration. We suggest, therefore, that the conformational change that results in the smaller radius of gyration for the ternary complex is a hinge motion of the two lobes which produces a closing of the cleft between the two lobes. The apparent similarity of the ligand-induced change in phosphoglycerate kinase to the cleft closure in hexokinase suggests that this kind of conformational change may prove to be a rather general kinase phenomenon (Bennett, W.S., and Steitz T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852; Anderson, C.M., Zucker, F.H., and Steitz, T.A. (1979) Science 204, 375-380).

Adenosine Triphosphate↗
PubMed1979

Space-filling models of kinase clefts and conformation changes.

Space-filling models of yeast hexokinase, adenylate kinase, and phosphoglycerate kinase drawn by computer clearly portray the bilobal character of these phosphoryl transfer enzymes, and the deep cleft which is formed between the lobes. A dramatic conformational change occurs in hexokinase as glucose binds to the bottom of the cleft, which causes the two lobes of hexokinase to come together. A substrate-induced closing of the active site cleft is postulated to occur in other kinases as well. This change may provide a mechanism by which some of these enzymes reduce their inherent adenosine triphosphatase activity and could be a general requirement of the kinase reaction.

Adenylate Kinase↗
PubMed1979

Small angle X-ray scattering of dimeric yeast hexokinase in solution.

Small angle x-ray scattering measurements on dimeric yeast hexokinase B at pH 5.5 in acetate buffer yield a radius of gyration of 31.28 +/- 0.23 angstrom. This measured value is comparable to the radius of gyration of 31.5 angstrom calculated from the refined coordinates of the dimer in the BII crystal form. The hexokinase dimer found in the BI crystal form has a radius of gyration of 42 angstrom calculated from the atomic coordinates. Thus, the measured radius of gyration is consistent with the BII dimer being the predominant species in solution and rules out the existence of the BI dimer as a major species under these conditions.

Hexokinase↗