Hongyuan Mao

The solution structure of a human immunodeficiency virus type-1 (HIV-1) Rev peptide bound to stem-loop IIB of the Rev response element (RRE) RNA was solved by nuclear magnetic resonance spectroscopy. The Rev peptide has an alpha-helical conformation and binds in the major groove of the RNA near a purine-rich internal loop. Several arginine side chains make base-specific contacts, and an asparagine residue contacts a G.A base pair. The phosphate backbone adjacent to a G.G base pair adopts an unusual structure that allows the peptide to access a widened major groove. The structure formed by the two purine-purine base pairs of the RRE creates a distinctive binding pocket that the peptide can use for specific recognition.

Sortase (SrtA), a transpeptidase from Staphylococcus aureus, catalyzes a cell-wall sorting reaction at an LPXTG motif by cleaving between threonine and glycine and subsequently joining the carboxyl group of threonine to an amino group of pentaglycine on the cell wall peptidoglycan. We have applied this transpeptidyl activity of sortase to in vitro protein ligation. We found that in the presence of sortase, protein/peptide with an LPXTG motif can be specifically ligated to an aminoglycine protein/peptide via an amide bond. Additionally, sortase can even conjugate substrates such as (d)-peptides, synthetic branched peptides, and aminoglycine-derivatized small molecules to the C terminus of a recombinant protein. The sortase-mediate protein ligation is robust, specific, and easy to perform, and can be widely applied to specific protein conjugation with polypeptides or molecules of unique biochemical and biophysical properties.

Many lead compounds bind to serum albumin and exhibit markedly reduced efficacy in vivo as compared to their potency in vitro. To aid in the design of compounds with reduced albumin binding, we performed nuclear magnetic resonance (NMR) structural and binding studies on the complex between domain III of human serum albumin (HSA-III) and diflunisal, a cyclooxygenase inhibitor with antiinflammatory activity. The structural studies indicate that the aromatic rings of diflunisal are involved in extensive and specific interactions with hydrophobic residues that comprise the binding pocket in subdomain IIIA. The carboxylic acid of diflunisal forms electrostatic interactions with the protein similar to those observed in the X-ray structure of HSA complexed to myristic acid. In addition to the structural studies, NMR-derived binding constants were obtained for diflunisal and closely related analogues to develop a structure-affinity relationship for binding to subdomain IIIA. On the basis of the structural and binding data, compounds were synthesized that exhibit more than a 100-fold reduction in binding to domain III of HSA, and nearly a 10-fold reduction in affinity for full length albumin. Significantly, several of these compounds maintain activity against cyclooxygenase-2. These results suggest a rational strategy for designing out albumin binding in potential drug molecules by using structure-based design in conjunction with NMR-based screening.