Hugh B. Nicholas

Reevaluation of the determinants of tyrosine sulfation.

The posttranslational sulfation of tyrosine has been though to be initiated by the recognition of specific consensus features by the sulfating enzyme tyrosylprotein sulfotransferase (TPST). However, using these recognition features to identify new tyrosine sulfation sites misses recently characterized sites that lack these features. Rigorous analysis of the amino acids surrounding the target tyrosin using the position-specific scoring matrix (PSSM) demonstrates that a consensus sequence does not contain all the information necessary to predict tyrosine sulfation. Instead, accurate prediction requires consideration of all residues within five amino acids on either side of the target tyrosine. These results support the notion that secondary structure is the major determinant of sulfation and that other residues within the sulfation site can compensate for deviations from commonly observed features. This view implies that specific consensus features are not critical for TPST substrate recognition but that TPST may instead broadly recognize any sufficiently exposed tyrosine residue.

Prediction of tyrosine sulfation in seven-transmembrane peptide receptors

Posttranslational modification by tyrosine sulfation regulates many important protein-protein interactions and modulates the binding affinity and specificity of seven-transmembrane peptide receptors. We developed a log-odds position-specific-scoring-matrix (PSSM) to accurately predict tyrosine sulfation using 62 tyrosine sites known to be sulfated and 421 tyrosine sites known not to be sulfated. We predict that 49 tyrosines of 32 seven-transmembrane peptide receptors are sulfated. Although we did not incorporate characteristics of confirmed sulfation sites such as clustering and conservation across species into our PSSM, our predicted sites nevertheless exhibited these characteristics. The observed conservation suggests that there are strong evolutionary pressures to preserve selected biological activity of seven-transmembrane receptors. The predicted tyrosine sulfation sites predominantly occur in the extracellular tail and extracellular loop 2, regions consistent with their association with binding pockets of the receptor.

Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation

Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near‐attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM‐Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM‐GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step‐wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process. Proteins 2004.

The Class D β-lactamase family: residues governing the maintenance and diversity of function

Class D β-lactamases, a major source of bacterial resistance to β-lactam antibiotic therapies, represent a distinct subset of the β-lactamase superfamily. They share a serine hydrolase mechanism with Classes A/C vs. Class B. Further understanding of their sequence-structure-function relationships would benefit efforts to design a new generation of antibiotics as well as to predict evolutionary mechanisms in response to such therapies. Here we describe analyses based on our high-resolution multiple sequence alignment and phylogenetic tree of ∼80 Class D β-lactamases that leverage several 3D structures of these enzymes. We observe several sequence clusters on the phylogenetic tree, some that are species specific while others include several species from α-, β- and γ-proteobacteria. Residues characteristic of a specific cluster were identified and shown to be located just outside the active site, possibly modulating the function of the catalytic residues to facilitate reactions with specific types of β-lactams. Most significant was the discovery of a likely disulfide bond in a large group composed of α-, β- and γ-proteobacteria that would contribute to enzyme stability and hence bacterial viability under antibiotic assault. A network of co-evolving residues was identified which suggested the importance of maintaining a surface for binding a highly conserved Phe69.

Interacting RNA species identified by combinatorial selection.

RNA molecules were selected from a random sequence library for their ability to bind to an RNA stem-loop target. Oligonucleotides with extensive Watson-Crick complementarity to the RNA ligand were selected against by inclusion of a blocking oligodeoxynucleotide in the binding phase of the selection protocol. After 18 generations of SELEX (systematic evolution of ligands by exponential enrichment) a single RNA family was predominant in the binding population. The winning aptamer RNA bound the target RNA with an apparent Kd = 70 nM. Structural mapping and Fe(II)-EDTA protection indicated that the target RNA interacted with small unpaired loops in the aptamer structure.

Distributing the comparison of DNA and protein sequences across heterogeneous supercomputers

No abstract available

Initial catalytic events in class 3 aldehyde dehydrogenase: MM and QM/MM simulations

A novel enzyme mechanism has been predicted by computer simulations for formation of the thiohemiacetal intermediate in the rat ALDH3A1 enzyme. We used molecular mechanics simulations to study the atomic details of substrate binding and quantum mechanical/molecular mechanical methods to study the Cys-243 thiolate attack on benzaldehyde (BA) substrate. BA was found to produce more reactive conformers when aligned for formation of the tetrahedral thiohemiacetal in the R-configuration. In addition, the sulfhydryl proton was seen to be important for initial binding of the substrate. Finally, the free energy differences between forming a thiohemiacetal oxyanion intermediate versus forming a neutral thiohemiacetal intermediate where a proton is donated to the intermediate from the surroundings strongly favor the latter. Our results suggest that the proton donor is the amide proton from the Cys-243 backbone supported by interactions with Lys-235.

MPI-PHYLIP: Parallelizing Computationally Intensive Phylogenetic Analysis Routines for the Analysis of Large Protein Families

Background Phylogenetic study of protein sequences provides unique and valuable insights into the molecular and genetic basis of important medical and epidemiological problems as well as insights about the origins and development of physiological features in present day organisms. Consensus phylogenies based on the bootstrap and other resampling methods play a crucial part in analyzing the robustness of the trees produced for these analyses. Methodology Our focus was to increase the number of bootstrap replications that can be performed on large protein datasets using the maximum parsimony, distance matrix, and maximum likelihood methods. We have modified the PHYLIP package using MPI to enable large-scale phylogenetic study of protein sequences, using a statistically robust number of bootstrapped datasets, to be performed in a moderate amount of time. This paper discusses the methodology used to parallelize the PHYLIP programs and reports the performance of the parallel PHYLIP programs that are relevant to the study of protein evolution on several protein datasets. Conclusions Calculations that currently take a few days on a state of the art desktop workstation are reduced to calculations that can be performed over lunchtime on a modern parallel computer. Of the three protein methods tested, the maximum likelihood method scales the best, followed by the distance method, and then the maximum parsimony method. However, the maximum likelihood method requires significant memory resources, which limits its application to more moderately sized protein datasets.

An algorithm for identification and ranking of family-specific residues, applied to the ALDH3 family

An algorithm for detecting amino acid residues characteristic of individual protein families from within aligned collections of paralogous sequences, and its application to the ALDH3 family versus the rest of the ALDH extended family is described. Residues illuminated by this analysis include a key intramolecular tether, a lysine that makes an intersubunit contact at the dimer interface, three residues in close association with the substrate-binding funnel, and a pair of residues suggested to participate in proton relay during the catalytic cycle.

Beyond the catalytic core of ALDH: a web of important residues begins to emerge

Site-directed mutagenesis was performed in class 3 aldehyde dehydrogenase (ALDH) on both strictly conserved, non-glycine residues, Glu-333 and Phe-335. Both lie in Motif 8 and are indicated to be of central catalytic importance from their positions in the tertiary structure. In addition, a highly conserved residue at the end of Motif 8, Pro-337, and Asp-247, which interacts with the main chain of Motif 8, were also mutated. All substitutions were conservative. Kinetic values clearly show that Glu-333 and Phe-335 are crucial to efficient catalysis, along with Asp-247. Pro-337 appears to have a different role, most likely relating to folding.