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M R Pincus

Publications and source records attributed to M R Pincus.

At least 91 records · Page 5Linked to original sources
PubMed1989

Comparative X-ray crystallographic evidence for a beta-bend conformation as the active structure for peptide T in T4 receptor recognition.

A sequence similarity has been found between two segments of endothiapepsin (acid proteinase, 2APE), bovine pancreatic ribonuclease A, and peptide T, a segment of the gp120 protein of human immune deficiency virus (HIV), which has been implicated in blocking viral attachment to the T4 receptor. The two similar sequences of the acid proteinase enzyme are Leu-Ile-Asp-Ser-Ser-Ala-Tyr-Thr (residues 169-176) and Tyr-Thr-Gly-Ser-Leu-Asn-Tyr-Thr (residues 175-182). Since the X-ray crystallographic structures of the acid proteinase and ribonuclease are known, it has been possible to determine whether the three-dimensional structures of the segments are similar. Portions of both the segments of acid proteinase are directly superimposable on the structure of the RNase A 19-26 segment. The fact that the three similar sequences from two completely unrelated proteins give rise to almost identical structures raises the possibility that these segments may be involved in nucleating the folding of these proteins. In addition, this provides further support for the concept that the octapeptide sequence of peptide T of HIV, which is also similar in sequence to the 19-26 sequence of RNase A, is also structurally similar to these residues, which adopt a beta-bend conformation. Furthermore, comparison of similarities and differences in the structure of these similar sequences provides an explanation for alterations in the biological activity of various truncated or substituted derivatives of peptide T and additional confirmation of the structural requirements for peptide T in T4-receptor recognition.

Animals↗
PubMed1989

Conformation of the metastasis-inhibiting laminin pentapeptide.

The binding of cancer cells to the basement membrane glycoprotein laminin appears to be a critical step in the metastatic process. This binding can be inhibited competitively by a specific pentapeptide sequence (Tyr-Ile-Gly-Ser-Arg) of the laminin B1 chain, and this peptide can prevent metastasis formation in vivo. However, other similar pentapeptide sequences (e.g., Tyr-Ile-Gly-Ser-Glu) have been found to be much less active in metastasis inhibition, raising the possibility that such amino acid substitutions produce structural changes responsible for altering binding to the laminin receptor. In this study, conformational energy analysis has been used to determine the three-dimensional structures of these peptides. The results indicate that the substitution of Glu for the terminal Arg produces a significant conformational change in the peptide backbone at the middle Gly residue. These results have important implications for the design of drugs that may be useful in preventing metastasis formation and tumor spread.

Amino Acids↗
PubMed1989

Conformational energy analysis of the leucine repeat regions of C/EBP, GCN4, and the proteins of the myc, jun, and fos oncogenes.

It has been recently proposed that certain DNA binding proteins (including C/EBP, GCN4 and the myc, jun, and fos oncogene proteins) share a common structural motif based on helix-promoting regions containing heptad repeat sequences of leucines. It has been suggested that this structure is critical to the biological activity of these proteins, since it facilitates the formation of functional dimers held together by interdigitating leucine side-chains along the hydrophobic interfaces between long alpha-helical regions of the polypeptide chains in a configuration termed the "leucine zipper." In this paper, conformational energy analysis is used to deterrmine the preferred three-dimensional structures of the leucine repeat regions of these proteins. The results indicate that, in all cases, the global minimum energy conformation for these regions is an amphipathic alpha-helix with the leucine side-chains arrayed on one side in such a way to favor "leucine zipper" dimerization. Furthermore, amino acid substitutions in these regions (such as Pro for Leu), that are known to inhibit dimer formation and prevent DNA binding, are found to produce significant conformational changes that disrupt the amphipathic helical structure. Thus, these results provide support for the proposed "leucine zipper" configuration as a critical structural feature of this class of DNA binding proteins.

Amino Acid Sequence↗
PubMed1989

Conformational effects of amino acid substitutions in the P-glycoprotein of the mdr 1 gene.

The P-glycoprotein of the mdr 1 gene is responsible for the phenomenon of multidrug resistance in human cells. The presumed drug-binding site of the wild-type P-glycoprotein contains a glycine at position 185. A mutant P-glycoprotein which contains valine at this position causes cells to retain resistance to colchicine, but to lose cross-resistance to other drugs such as the chemotherapeutic agents vinblastine and Adriamycin. This has been hypothesized to be due to a conformational change in the protein induced by the amino acid substitution. Using conformational energy analysis, we have determined the allowed three-dimensional structures for the wild-type and mutant proteins in the region of position 185. The results indicate that the wild-type protein adopts a unique left-handed conformation at position 185 which is energically unfavorable for the protein with L-amino acids (including valine) at this position. This conformational change induced by amino acid substitutions for Gly 185 could explain the differences in binding to the P-glycoprotein of various drugs and, hence, the differences in drug resistance exhibited by various cell lines expressing these proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

Affinity Labels↗
PubMed1989

Comparison of the computed structures for the phosphate-binding loop of the p21 protein containing the oncogenic site Gly 12 with the X-ray crystallographic structures for this region in the p21 protein and EFtu. A model for the structure of the p21 protein in its oncogenic form.

The GTP-binding p21 protein encoded by the ras-oncogene can be activated to cause malignant transformation of cells by substitution of a single amino acid at critical positions along the polypeptide chain. Substitution of any non-cyclic L-amino acid for Gly 12 in the normal protein results in a transforming protein. This substitution occurs in a hydrophobic sequence (residues 6-15) which is known to be involved in binding the phosphate moities of GTP (and GDP). We find, using conformational energy calculations, that the 6-15 segment of the normal protein (with Gly 12) adopts structures that contain a bend at residues 11 and 12 with the Gly in the D* conformation, not allowed energetically for L-amino acids. Substitution of non-cyclic L-amino acids for Gly 12 results in shifting this bend to residues 12 and 13. We show that many computed structures for the Gly 12-containing phosphate binding loop, segment 9-15, are superimposable on the corresponding segment of the recently determined X-ray crystallographic structure for residues 1-171 of the p21 protein. All such structures contain bends at residues 11 and 12 and most of these contain Gly 12 in the C* or D* conformational state. Other computed conformations for the 9-15 segment were superimposable on the structure of the corresponding 18-23 segment of EFtu, the bacterial chain elongation factor having structural similarities to the p21 protein in the phosphate-binding regions. This segment contains a Val residue where a Gly occurs in the p21 protein. As previously predicted, all of these superimposable conformations contain a bend at positions 12 and 13, not 11 and 12. If these structures that are superimposable on EFtu are introduced into the p21 protein structure, bad contacts occur between the sidechain of the residue (here Val) at position 12 and another phosphate binding loop region around position 61. These bad contacts between the two segments can be removed by changing the conformation of the 61 region in the p21 protein to the corresponding position of the homologous region in EFtu. In this new conformation, a large site becomes available for the binding of phosphate residues. In addition, such phenomena as autophosphorylation of the p21 protein by GTP can be explained with this new model structure for the activated protein which cannot be explained by the structure for the non-activated protein.

Cell Transformation, Neoplastic↗
PubMed1989

Correlation of beta-bend conformations of tetrapeptides with their activities in CD4-receptor binding assays.

Conformational analysis, based on ECEPP (Empirical Conformational Energy Program for Peptides) using the chain build-up procedure, was applied to determine the low-energy conformations for a series of tetrapeptides. The tetrapeptides are components of larger peptides which have been found to bind to the CD4 receptor of monocytes. Several previous studies have implicated the tetrapeptide units investigated here as being critical to the biological activities of the full peptides. Five such tetrapeptides were studied: Ser-Ser-Asn-Tyr (from ribonuclease A), Thr-Thr-Asn-Tyr (from peptide T, known to block human immunodeficiency virus from attaching to CD4+ T cells), Thr-Ile-Asn-Tyr (from polio virus coat protein, which is less active than the other peptides in binding to CD4 receptors), Ser-Ser-Ala-Tyr (from the gp 120 coat protein of human immunodeficiency virus, a variant of the peptide T sequence, active in blocking viral attachment to CD4+ cells), and the tetrapeptide from an active synthetic pentapeptide, Asn-Thr-Lys-Tyr (from Asn-Thr-Lys-Tyr-Thr). Using a 7 kcal/mol cutoff, the low-energy conformations for each peptide were computed. Approximately 20,000 conformations were computed for each tetrapeptide. Residue probability profiles were determined for each tetrapeptide. All tetrapeptides except for the polio sequence showed flexibility in the sense that many low-energy conformations were possible. In previous studies, it was postulated that the critical tetrapeptide units would adopt conformations similar to the one observed in a segment of ribonuclease A, residues 22-25, a beta-bend, which is part of an octapeptide segment (residues 19-26) that is homologous to the sequence of peptide T.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

The structure of the amino terminal transforming segment of the p21 protein, Tyr4-Thr20 (with Asp12), by two-dimensional NMR.

The structure of a peptide from the transforming region (residues 4-20) of the p21 protein has been determined using two-dimensional NMR. In the normal protein, this segment contains a Gly residue at the critical 12 position; any substitution, other than Pro, at this position results in a transforming protein. Previously performed energy calculations indicated that this peptide segment is a structured one. In this study we find that the Asp12 containing peptide has a surprisingly well-defined structure in solution which has more similarity to the GDP-binding loop region in EF-tu than to that in p21.

Amino Acid Sequence↗
PubMed1988

Conformational effects of the substitution of Arg for Gly 13 in the ras oncogene-encoded P21 protein.

The effect of the substitution of Arg for Gly 13 on the structure of the transforming region decapeptide (Leu 6-Gly 15) of the ras oncogene encoded P21 protein has been investigated using conformational energy analysis. A human malignancy has been identified that contains a ras gene with a single mutation in the thirteenth codon such that the encoded protein would have Arg substituted for Gly at this position, and transfection of cells in culture with this gene results in malignant transformation. Conformational analysis demonstrates that the Arg 13 decapeptide adopts a conformation identical to that for other peptides with substitutions at position 13 (Asp 13, Val 13) from transforming proteins that is distinctively different from that for peptides (Gly 13, Ser 13) from normal, nontransforming proteins. This is found to be an indirect effect resulting from changes in the conformation of Gly 12 produced by substitutions at position 13. These results are consistent with recent analysis of crystallographic data of proteins on conformational preferences for glycine in tripeptide sequences.

Arginine↗
PubMed1988

Conformational analysis of possible biologically active (receptor-bound) conformations of peptides derived from cholecystokinin, cerulein and little gastrin and the opiate peptide, Met-enkephalin.

Possible biologically active (receptor-bound) conformations of peptides derived from cholecystokinin (CCK) have been deduced using conformational analysis combined with comparative studies of their biological specificities. Two peptides, the completely active carboxyl terminal heptapeptide from CCK (CCK-7), whose sequence is Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, and the carboxyl terminal heptapeptide from cerulein (CER-7) which has the same sequence as for CCK-7 except for replacement of Met 2 with a Thr 2, both stimulate peripheral receptors in gall bladder, pancreas, and pylorus in the gastrointestinal system. In contrast, two other very similar peptides, the last four residues of CCK (CCK-4) whose sequence is Trp-Met-Asp-Phe-NH2, and the carboxyl terminal hexapeptide of little gastrin (LGA-6, Tyr-Gly-Trp-Met-Asp-Phe-NH2, i.e., residue 2 deleted relative to CCK-7 and CER-7 sequences), interact specifically with gastrin receptors and not at all or very weakly with peripheral receptors. All of these peptides react with CCK receptors in the central nervous system, especially in forebrain. The results in the GI tract suggest that the peptides active on peripheral receptors adopt structures that are significantly different from those of the peptides that interact with gastrin receptors. We have generated all of the many low energy conformations for each of these peptides. By retaining only the conformations that are the same for peptides within the same group and then rejecting those resulting conformations that are the same for the peptides in the two different groups, we can greatly reduce the possible active conformations for the peptides within each class.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Structure of the carboxyl terminus of the RAS gene-encoded P21 proteins.

The three-dimensional structures of the carboxyl-terminal regions of the P21 protein products of the human Harvey (Ha), Kirsten (KiA and KiB), and neuroblastoma (N) RAS oncogenes and various mutants have been determined by using conformational energy analysis. The carboxyl-terminal region of P21 has been strongly implicated in the binding of the protein to the inner surface of the plasma membrane without which the protein is inactive. The only invariant residue in this region is Cys-186, which is necessary for the post-translational addition of palmitic acid. The surrounding sequences of the active native proteins differ considerably. Nevertheless, certain amino acid substitutions in this region are known to eliminate membrane binding and protein activity, suggesting that there is a conserved common structural feature in this region in the native proteins that is disrupted in the mutant proteins. Conformational energy analysis shows that the four native P21 proteins have a common structure in the form of an alpha-helix for the terminal pentapeptide. A mutant, pBW277, that fails to bind to the membrane and is inactive cannot adopt an alpha-helical structure in this region because of a proline at position 188. Another mutant, pBW766, that retains membrane binding and activity, on the other hand, retains the preference for an alpha-helical conformation in the terminal pentapeptide. These findings suggest that, despite various amino acid sequences in this region, the carboxyl-terminal pentapeptides of the P21 proteins form a distinctive structural domain that must have an alpha-helical structure for membrane binding and intracellular activity.

Cell Membrane↗
PubMed1988

Use of creatine kinase MB isoenzyme for diagnosing myocardial infarction when total creatine kinase activity is high.

The usefulness of measuring creatine kinase MB isoenzyme for diagnosing myocardial infarction when activities of total creatine kinase are very high is unclear. We conducted a retrospective study in an urban hospital that serves a largely indigent population. We concentrated on 146 patients whose creatine kinase activity was greater than 1000 U/L (upper limit of normal: 165 U/L for women and 225 U/L for men), with MB isoenzyme greater than 10 U/L and less than 5% of total creatine kinase. The positive predictive value of MB isoenzyme (isoimmune method) values greater than 10 U/L was between 11.6% and 56.8% when the value for total creatine kinase exceeded 1000 U/L. Using different values (MB greater than 4% of total creatine kinase) as positive for myocardial infarction would have resulted in far fewer false-positives, but 10 cases of myocardial infarction would have been missed. The most appropriate cutoff value for MB isoenzyme in this population (total creatine kinase greater than 1000 U/L) was found to be greater than 2% of total creatine kinase.

Adolescent↗
PubMed1987

A strong homology exists between the active T-cell binding gp120 octapeptide of human immunodeficiency virus and the subtilisin cleavage peptide of bovine ribonuclease A.

A homology has been found between an octapeptide involved in attachment of the human immunodeficiency virus to helper/inducer T cells and an octapeptide segment of bovine pancreatic ribonuclease A. This segment (residues 19-26) contains the sites for subtilisin cleavage of this enzyme into the S-peptide and S-protein. From the X-ray crystal structure of ribonuclease, this sequence is known to be exposed to solvent and interacts little with the rest of the protein. A structure for the human immunodeficiency virus attachment peptide can be deduced from this homology, as a well-defined structure has been determined for this sequence in ribonuclease. This can be readily accomplished using previously developed computer methods based upon conformational energy calculations. The calculated structure for human immunodeficiency virus peptide is identical to the ribonuclease segment (19-26) in backbone conformation. It is stabilized by internal interactions of nonpolar residues, and by exposure of polar hydroxyl groups. The results suggest that the T-cell human immunodeficiency virus receptor may be hydrophilic in nature and that conservation of the sequence in two presumably functionally unrelated proteins is related to the need for conservation of exposed structure.

Amino Acid Sequence↗
PubMed1987

A model for the three-dimensional structure of peptidoglycan in staphylococci.

Although the monomeric units of peptidoglycan in Staphylococcus aureus and other staphylococci are well known, the complete structure of the peptidoglycan has not been elucidated. The peptidoglycan monomeric unit may be divided into three parts: (1) glycan chain piece, consisting of N-acetylglucosaminyl-N-acetylmuramic acid; (2) connecting peptide extending from L-alanine to the alpha-amino group of L-lysine; (3) peptide chain piece, consisting of D-alanine, the remainder of L-lysine not included in the connecting peptide, and pentaglycine (S. aureus) or mixed glycine and serine residues (other staphylococci) attached to the epsilon amino group of lysine. The deformation of cross wall into hemisphere in the course of cell division, the distensibility of peptidoglycan, and the appearance of circular (? spiral) lines in the cross wall and on the surface of the newly-formed hemisphere are clues to the structure of peptidoglycan. In the proposed model, cross wall is formed as a linear spiral with 20 turns extending in a plane from periphery to center of the cell. During cell division, the cross wall is bisected. The cross wall spiral becomes a spiral forming the peripheral wall of a new hemisphere. The width of the spiral on the cell surface is maintained by rigid glycan chains and by covalent bonds linking turns of the spiral. The length of the spiral is about 30 times the diameter of the cell. Flexible polypeptide sheets consisting of parallel polypeptide chains run along the length of the spiral. Individual polypeptides contain an average of ten peptide chain pieces. The glycan chain is a helix with two disaccharide residues per turn; consequently consecutive connecting peptides project in opposite directions and are perpendicular both to the glycan chain and to the peptide chain. In cross wall, hydrogen bonding between polypeptide chains enables the polypeptide sheet to transmit changes in tension. The deformation of cross wall into peripheral wall requires doubling of the external surface area of the peptidoglycan. A change in the angle of the glycan chain with respect to the peptide chain results in an increase of the distance between peptide chains, causing the doubling of surface area. Implications of the model include explanations for the initiation of cell division and for the existence of osmotically growth-dependent staphylococci.

Cell Division↗
PubMed1987

Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rate.

Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3 have been determined for each derivative. They are the following: 3'-(bromoacetamido)-3'-deoxyuridine, Kb = 0.062 M, k3 = 3.3 X 10(-4) s-1; 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 X 10(-4) s-1; 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 X 10(-4) s-1; and 3'-(bromoacetamido)-3'-deoxythymidine, Kb = 0.094 M, k3 = 2.7 X 10(-4) s-1. 3'-(Bromoacetamido)-3'-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil and 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3'-(Bromoacetamido)-3'-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 of histidine-119. A specific interaction of the "down" 2'-OH group, unique to 3'-(bromoacetamido)-3'-deoxyuridine, serves to orient the 3'-bromoacetamido residue close to the imidazole ring of histidine-119. The 2'-OH group of 3',5'-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.

Acetamides↗
PubMed1987

On the biologically active structures of cholecystokinin, little gastrin, and enkephalin in the gastrointestinal system.

The biologically active conformations of a series of four peptides [four cholecystokinin (CCK)-related peptides and enkephalin] in their interactions with gastrointestinal receptors have been deduced using conformational computational analysis. The two peptides that interact exclusively with peripheral-type CCK receptors are the heptapeptide COOH-terminal fragment from CCK (CCK-7) and the analogous sequence from cerulein (CER-7) in which threonine replaces the methionine proximal to the NH2 terminus. The two peptides that interact exclusively with the gastrin receptor in the stomach are the active COOH-terminal fragment of little gastrin and the COOH-terminal tetrapeptide sequence common to all of these peptides, CCK-4. We find that preferred conformations for the peripherally active peptides CCK-7 and CER-7 are principally beta-bends, whereas little gastrin and CCK-4 are fundamentally helical. In the class of lowest energy structures for both CCK-7 and CER-7, the aromatic rings of the tyrosine and phenylalanine lie close to one another whereas the tryptophan indole ring points in the opposite direction. This structure is superimposable on the structures of a set of rigid indolyl benzodiazepine derivatives that interact with complete specificity and high affinity with peripheral CCK receptors further suggesting that the computed beta-bends are the biologically active conformation. The biologically active conformation for CCK-4 and the little gastrin hexapeptide has also been deduced. By excluding conformations common to CCK-7 and CCK-4, which do not bond to each other's receptors, and then by selecting conformations in common to CCK-4 and the gastrin-related hexapeptide, which do bind to each other's receptors, we deduce that the biologically active conformation at the gastrin receptor is partly helical and one in which the indole of tryptophan and the aromatic ring of phenylalanine are close to one another while the methionine and aspartic acid side chains point in the opposite direction. These major differences in preferred structures between the common CCK-7/CER-7 peptides and the common CCK-4/little gastrin peptides explain the mutually exclusive activities of these two sets of peptides. We have observed that [Met]enkephalin strongly antagonizes the action of the naturally occurring peripherally active CCK-8 (CCK-7 with an NH2-terminal aspartic acid residue added). The computed lowest energy structures for this opiate peptide closely resemble key features of the computed CCK-7/CER-7 structure, further supporting the proposed structure.

Amino Acid Sequence↗