Intrinsic flexibility of West Nile virus protease in solution characterized using small-angle X-ray scattering.

West Nile virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection can cause severe neurological disease and fatality in humans. Efforts are ongoing to develop antiviral drugs that inhibit the WNV protease, a viral enzyme required for polyprotein processing. Unfortunately, little is known about the solution structure of recombinant WNV protease (NS2B-NS3pro) used for antiviral drug discovery and development, although X-ray crystal structures and nuclear magnetic resonance (NMR) studies have provided valuable insights into the interactions between NS2B-NS3pro and peptide-based inhibitors. We completed small-angle X-ray scattering and Fourier transform infrared spectroscopy experiments to determine the solution structure and dynamics of WNV NS2B-NS3pro in the absence of a bound substrate or inhibitor. Importantly, these solution studies suggested that all or most of the NS2B cofactor was highly flexible and formed an ensemble of structures, in contrast to the NS2B tertiary structures observed in crystallographic and NMR studies. The secondary structure of NS2B-NS3pro in solution had high ß-content, similar to the secondary structure observed in crystallographic studies. This work provided evidence of the intrinsic flexibility and conformational heterogeneity of the NS2B chain of the WNV protease in the absence of substratelike ligands, which should be considered during antiviral drug discovery and development efforts.

Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis.

FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains. The unfolding/folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions.