Biomedical subjects

David F Green

Publications and source records attributed to David F Green.

5 recordsLinked to original sources

PubMed2006

Rational design of new binding specificity by simultaneous mutagenesis of calmodulin and a target peptide.

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies the role of CaM as a shared participant in calcium-dependent signal transduction pathways but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/target pairs that retain high affinity for one another but lack affinity for wild-type CaM and its natural interaction partners would therefore be useful as sensor components and possibly also as elements of "synthetic" cellular-signaling networks. Here, we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal-muscle light-chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained an affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple-linked binding equilibria.

Amino Acid Sequence↗

PubMed2005

Design of improved protein inhibitors of HIV-1 cell entry: Optimization of electrostatic interactions at the binding interface.

Continuum electrostatic methods are a powerful tool for the analysis and design of biomolecular complexes, with methodologies that allow for the detailed analysis of the electrostatic contributions to binding affinities and procedures for computing the properties of electrostatically optimal ligands. We have applied these methods to the design of improved inhibitors of HIV-1 cell entry. HIV infection of a cell requires viral-cell membrane fusion, an event partially driven by a large-scale conformational change in the viral membrane glycoprotein gp41. This transformation involves the docking of a helix from the C-terminal region of three gp41 chains against a pre-formed trimeric-coiled coil; several protein constructs that inhibit membrane fusion act by binding to an isolated C-terminal helix and blocking the formation of the fusogenic structure. A detailed analysis of the electrostatic contributions to the binding of one such inhibitor (5-Helix) to a C-terminal helix was performed using the X-ray crystal structure of the core of the HIV-1 gp41 ectodomain as a structural model, and several residues on 5-Helix that make substantial contributions to binding, both favorable and unfavorable, were identified. An electrostatic affinity optimization methodology was applied to the side chains of 5-Helix, with the results showing that significant improvements in binding affinity are possible if the electrostatic contributions to the binding free energy are optimized. Several mutations accessible by experimental methods are suggested, with calculated improvements in binding affinity of as much as 500-fold and greater.

Amino Acid Sequence↗

PubMed2005

Action-at-a-distance interactions enhance protein binding affinity.

The identification of protein mutations that enhance binding affinity may be achieved by computational or experimental means, or by a combination of the two. Sources of affinity enhancement may include improvements to the net balance of binding interactions of residues forming intermolecular contacts at the binding interface, such as packing and hydrogen-bonding interactions. Here we identify noncontacting residues that make substantial contributions to binding affinity and that also provide opportunities for mutations that increase binding affinity of the TEM1 beta-lactamase (TEM1) to the beta-lactamase inhibitor protein (BLIP). A region of BLIP not on the direct TEM1-binding surface was identified for which changes in net charge result in particularly large increases in computed binding affinity. Some mutations to the region have previously been characterized, and our results are in good correspondence with this results of that study. In addition, we propose novel mutations to BLIP that were computed to improve binding significantly without contacting TEM1 directly. This class of noncontacting electrostatic interactions could have general utility in the design and tuning of binding interactions.

Hydrogen Bonding↗

PubMed2004

Escherichia coli glutaminyl-tRNA synthetase is electrostatically optimized for binding of its cognate substrates.

Natural evolution has resulted in protein molecules displaying a wide range of binding properties that include extremes of affinity and specificity. A detailed understanding of the principles underlying protein structure-function relationships, particularly with respect to binding properties, would greatly enhance molecular engineering and ligand design studies. Here, we have analyzed the interactions of an aminoacyl-tRNA synthetase for which strong evolutionary pressure has enforced high specificity for substrate binding and catalysis. Electrostatic interactions have been identified as one efficient mechanism for enhancing binding specificity; as such, the effects of charged and polar groups were the focus of this study. The binding of glutaminyl-tRNA synthetase from Escherichia coli to several ligands, including the natural substrates, was analyzed. The electrostatic complementarity of the enzyme to its ligands was assessed using measures derived from affinity optimization theory. The results were independent of the details of the calculational parameters, including the value used for the protein dielectric constant. Glutamine and ATP, two of the natural ligands, were found to be extremely complementary to their binding sites, particularly in regions seen to make electrostatic interactions in the structure. These data suggest that the optimization of electrostatic interactions has played an important role in guiding the evolution of this enzyme. The results also show that the enzyme is able to effectively select for high affinity and specificity for the same chemical moieties both in the context of smaller substrates, and in that of a larger reactive intermediate. The regions of greatest non-complementarity between the enzyme and ligands are the portions of the ligand that make few polar contacts with the binding site, as well as the sites of chemical reaction, where overly strong electrostatic binding interactions with the substrate could hinder catalysis. The results also suggest that the negative charge on the phosphorus center of glutaminyl-adenylate plays an important role in the tight binding of this intermediate, and thus that adenylate analogs that preserve the negative charge in this region may bind substantially tighter than analogs where this group is replaced with a neutral group, such as the sulfamoyl family, which can make similar hydrogen bonds but is uncharged.

Adenosine Monophosphate↗

PubMed2004

X-ray structural and simulation analysis of a protein mutant: the value of a combined approach.

The effect of the mutation Arg 96 to His on the stability of bacteriophage T4 lysozyme has been previously studied by calorimetric experiments, X-ray crystallography, and free energy simulation techniques. The experimental and calculated values for the difference between the free energy of denaturation of the mutant and the wild type are in reasonable agreement. However, the two approaches led to different explanations for the loss in stability. To analyze the differences, a series of refinements based on the crystallographic data were performed, a number of aspects of the simulations were reexamined, and continuum electrostatic calculations were done to complement the latter. The results of those comparisons provide a better understanding of the origin of the free energy difference in this mutant. Furthermore, they show the importance of the combined use of simulations and crystallography for interpreting the effects of mutations on the energetics of the system.

Bacteriophage T4↗