Dipolar pathways in dipolar EPR spectroscopy

Dipolar electron paramagnetic resonance (EPR) experiments such as double electron--electron resonance (DEER) measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization of proteins and...

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterized Ab1303 and Ab1573, neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding was observed only when Env trimers were not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures showed that both antibodies recognized the CD4bs on Env trimer with an occluded-open conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation included outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, did not exhibit V1V2 displacement, co-receptor binding site exposure, or a 4-stranded gp120 bridging sheet. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggested an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

Functional basis of electron transport within photosynthetic complex I

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme – the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.