Structure-based antibody design and accurate predictions of antibody-antigen interactions remain major challenges in computational biology. By using molecular dynamics simulations, we show that a single static X-ray structure is not sufficient to identify determinants of antibody-antigen recognition. Here, we investigate antibodies that undergo substantial conformational changes upon antigen binding and have been classified as difficult cases in an extensive benchmark for antibody-antigen docking. We present thermodynamics and transition kinetics of these conformational rearrangements and show that paratope states can be used to improve antibody-antigen docking. By using the unbound antibody X-ray structure as starting structure for molecular dynamics simulations, we retain a binding competent conformation substantially different to the unbound antibody X-ray structure. We also observe that the kinetically dominant antibody paratope conformations are chosen by the bound antigen conformation with the highest probability. Thus, we show that paratope states in solution can improve antibody-antigen docking and structure prediction.

Comment on Structure. 2022 Mar 3;30(3):360-370.e6.

Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the QN and QP sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme bL. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.

Comment in Structure. 2022 Mar 3;30(3):326-328.

Comment on Structure. 2022 Mar 3;30(3):350-359.e3.

Update of bioRxiv. 2021 Aug 04;:

Comment in Structure. 2022 Mar 3;30(3):321-323.

B cell lymphoma 6 (BCL6) is a transcriptional repressor that is deregulated in diffuse large B cell lymphoma, and the peptide aptamer, Apt48, inhibits BCL6 by an unknown mechanism. We report the crystal structure of BCL6 in complex with an Apt48 peptide, and show that Apt48 binds to a therapeutically uncharacterized region at the bottom of the BCL6 BTB domain. We show that the corepressor binding site of the BTB domain may be divided conceptually into two low-affinity, peptide-binding regions. An upper region, the lateral groove, binds peptides in robust three-dimensional conformations, whereas a lower binding site is permissive to less-specific interactions. We show that, even with little sequence specificity, the interactions of the lower region are required for the high-affinity binding of the SMRT corepressor and other peptides to the BTB domain. This has relevance for the design of new BCL6 inhibitors and for understanding the evolution of corepressor interactions with the BTB domain.

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A2AAR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A2AAR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A2AAR structures.

Cryoelectron tomography (cryo-ET) and subtomogram averaging (STA) allow direct visualization and structural studies of biological macromolecules in their native cellular environment, in situ. Often, low signal-to-noise ratios in tomograms, low particle abundance within the cell, and low throughput in typical cryo-ET workflows severely limit the obtainable structural information. To help mitigate these limitations, here we apply a compressed sensing approach using 3D second-order total variation (CS-TV2) to tomographic reconstruction. We show that CS-TV2 increases the signal-to-noise ratio in tomograms, enhancing direct visualization of macromolecules, while preserving high-resolution information up to the secondary structure level. We show that, particularly with small datasets, CS-TV2 allows improvement of the resolution of STA maps. We further demonstrate that the CS-TV2 algorithm is applicable to cellular specimens, leading to increased visibility of molecular detail within tomograms. This work highlights the potential of compressed sensing-based reconstruction algorithms for cryo-ET and in situ structural biology.

Rab9 is mainly located on late endosomes and required for their intracellular transport to trans-Golgi network (TGN). The cytoplasmic dynein motor, together with its regulatory proteins Nde1/Ndel1 and Lis1, controls intracellular retrograde transport of membranous organelles along the microtubule network. How late endosomes are tethered to the microtubule-based motor dynein for their retrograde transport remains unclear. Here, we demonstrate that the guanosine triphosphate (GTP)-bound Rab9A/B specifically uses Nde1/Ndel1 as an effector to interact with the dynein motor complex. We determined the crystal structure of Rab9A-GTP in complex with the Rab9-binding region of Nde1. The functional roles of key residues involved in the Rab9A-Nde1 interaction are verified using biochemical and cell biology assays. Rab9A mutants unable to bind to Nde1 also failed to associate with dynein, Lis1, and dynactin. Therefore, Nde1 is a Rab9 effector that tethers Rab9-associated late endosomes to the dynein motor for their retrograde transport to the TGN.

Comment in Structure. 2022 Mar 3;30(3):324-326.

Comment on Structure. 2022 Mar 3;30(3):371-385.e5.