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Mark D Distefano

Publications and source records attributed to Mark D Distefano.

7 recordsLinked to original sources
PubMed2003

Structure and function analysis of peptide antagonists of melanoma inhibitor of apoptosis (ML-IAP).

Melanoma inhibitor of apoptosis (ML-IAP) is a potent anti-apoptotic protein that is upregulated in a number of melanoma cell lines but not expressed in most normal adult tissues. Overexpression of IAP proteins, such as ML-IAP or the ubiquitously expressed X-chromosome-linked IAP (XIAP), in human cancers has been shown to suppress apoptosis induced by a variety of stimuli. Peptides based on the processed N-terminus of Smac/DIABLO can negate the ability of overexpressed ML-IAP or XIAP to suppress drug-induced apoptosis. Such peptides have been demonstrated to bind to the single baculovirus IAP repeat (BIR) of ML-IAP and the third BIR of XIAP with similar high affinities (approximately 0.5 microM). Herein, we use phage-display of naïve peptide libraries and synthetic peptides to investigate the peptide-binding properties of ML-IAP-BIR and XIAP-BIR3. X-ray crystal structures of ML-IAP-BIR in complex with Smac- and phage-derived peptides, together with peptide structure-activity-relationship data, indicate that the peptides can be modified to provide increased binding affinity and selectivity for ML-IAP-BIR relative to XIAP-BIR3. For instance, substitution of Pro3' in the Smac-based peptide (AVPIAQKSE) with (2S,3S)-3-methylpyrrolidine-2-carboxylic acid [(3S)-methyl-proline] results in a peptide with 7-fold greater affinity for ML-IAP-BIR and about 100-fold specificity for ML-IAP-BIR relative to XIAP-BIR3.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase.

Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein. The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets. While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood. To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods. Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM. Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase. However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate. Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site. Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein. Taken together, these data do not support the "molecular ruler hypotheses". Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.

Alkyl and Aryl Transferases↗
PubMed2003

Diazotrifluoropropionamido-containing prenylcysteines: syntheses and applications for studying isoprenoid-protein interactions.

Photoaffinity-labeled prenylcysteines (1 and 2) incorporating a diazotrifluoropropionamide-based photophore have been prepared. Photolyses of 2 in the presence of RhoGDI, a protein that interacts with prenylated proteins, and prenylcysteine-containing competitors demonstrate the effectiveness of this photoaffinity-labeled analogue as a tool for studying isoprenoid binding sites.

Amides↗
PubMed2003

Use of synthetic isoprenoid analogues for understanding protein prenyltransferase mechanism and structure.

Protein prenylation involves the post-translational modification of specific protein-derived cysteine residues with farnesyl or geranylgeranyl groups through thioether linkages. Because a large number of proteins that participate in signal transduction processes require this modification, there has been intense interest in developing inhibitors of these enzymes and in clarifying the biological function of prenylation. Isoprenoid analogues have proven to be versatile tools for probing the mechanism and structure of prenyltransferases. Mechanistic probes have been created to investigate the stereochemical course and substituent effects in prenyltransferase catalyzed reactions. They have also been used to measure kinetic isotope effects and search for possible cationic intermediates. Photoaffinity labeling analogues containing either diazotrifluoropropionate or benzophenone units have been used to identify the location of isoprenoid binding sites in these enzymes. Biophysical probes incorporating fluorescent moieties or isotopic labels have been used to measure isoprenoid dissociation constants or prenyl group conformation when bound to the enzyme. Analogues containing noncognate alkene isomers or bulky substituents have also contributed to an understanding of isoprenoid recognition. Most recently, photoactive and isomeric isoprenylated analogues are providing insights into the function of protein prenylation.

Benzophenones↗
PubMed2002

Quantifying beta-sheet stability by phage display.

The small immunoglobulin G (IgG)-binding protein GB1 is a favored model system for the study of individual residue contributions to the stability of beta-sheets. Nevertheless, only a few of the many possible combinations of mutations have been characterized, leaving many questions unanswered. In order to allow the simultaneous evaluation of libraries of mutants, we have adapted a phage-display method, called shotgun scanning. This method combines a binding (i.e. stability) selection with high-throughput sequence analysis. Relative folding free energies determined from GB1-phage sequence data agree well with published GB1 thermal stability studies, validating the use of phage display to conduct quantitative stability studies on GB1, and further suggesting that this method is generally applicable to mutational analysis of protein stability. Examination of residue pairing in our large collection of GB1 mutants indicates that specific side-chain-side-chain interactions are much less important to beta-sheet stability than individual residue contributions. The discrepancy between this observation and published studies can be traced to anomalous stability of the alanine-substituted GB1 variants typically used as reference states in double mutant-cycle analyses. Finally, the combination of large library sizes and a quantitative stability selection should allow phage-based "computation" to be applied to protein design problems.

Bacteriophages↗
PubMed2002

Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors.

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.

Amination↗