Gauging the Flexibility of the Active Site in Soybean Lipoxygenase‑1 (SLO-1) through an Atom-Centered Density Matrix Propagation (ADMP) Treatment That Facilitates the Sampling of Rare Events

We present a computational methodology to sample rare events in large biological enzymes that may involve electronically polarizing, reactive processes. The approach includes simultaneous dynamical treatment of electronic and nuclear degrees of freedom, where contributions from the electronic portion are computed using hybrid density functional theory and the computational costs are reduced through a hybrid quantum mechanics/molecular mechanics (QM/MM) treatment. Thus, the paper involves a QM/MM dynamical treatment of rare events. The method is applied to probe the effect of the active site elements on the critical hydrogen transfer step in the soybean lipoxygenase-1 (SLO-1) catalyzed oxidation of linoleic acid. It is found that the dynamical fluctuations and associated flexibility of the active site are critical toward maintaining the electrostatics in the regime where the reactive process can occur smoothly. Physical constraints enforced to limit the active site flexibility are akin to mutations and, in the cases studied, have a detrimental effect on the electrostatic fluctuations, thus adversely affecting the hydrogen transfer process

The Disulfide Bonds within BST‑2 Enhance Tensile Strength during Viral Tethering

Human BST-2/tetherin is a host factor that inhibits the release of enveloped viruses, including HIV-1, HIV-2, and SIV, from the cell surface by tethering viruses to the host cell membrane. BST-2 has an α-helical ectodomain that forms disulfide-linked dimers between two monomers forming a coiled coil. The ectodomain contains three cysteine residues that can participate in disulfide bond formation and are critical for viral tethering. The role of the disulfides in viral tethering is unknown but proposed to be for maintaining the dimer. We explored the role of the disulfides in the structure of BST-2 using experimental, biophysical methods. To understand the role of the disulfides in viral tethering, we used a new approach in viral tethering, steered molecular dynamics. We find that the disulfides coordinate the unfolding of the BST-2 monomers, which adds tensile strength to the coiled coil. Structural differences between oxidized and reduced BST-2 are apparent during unfolding, showing the monomers slide past each other in the absence of the disulfides. We found no evidence to support dissociation of the dimer upon reduction of the disulfide bonds. Moreover, the structure of BST-2 in the absence of the disulfides is similar to that of the oxidized form of BST-2, supporting previous X-ray crystallography and cellular work that showed the disulfides are not required for expression of BST-2. These data provide new insights into viral tethering by using novel techniques in the analysis of BST-2 to give amino acid level insight into functions of BST-2