Gruzinov, A. | Search bioRxiv

bioRxiv2025

Capturing transient states of heterodimeric ABC transporter TM287/288 by Time-Resolved Small-Angle X-ray Scattering

Structures of the heterodimeric ABC transporter TM287/288 have been previously determined in several states indicating large conformational changes during its reaction cycle. However, for a complete description of the cycle, transient states (such as an occluded state) are still missing, as they are difficult to capture with static structural biology techniques. Here, we investigate the kinetics and accompanying structural changes in the course of the reaction cycle of full-length TM287/288 using time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing. The use of active site mutants and state-specific sybodies/nanobodies enabled us to dissect the temporal events involved in the ATP-driven conformational cycle of TM287/288 and reveal a transient occluded state of this ABC transporter. Statement of SignificanceATP-binding cassette transporters are essential molecular machines that use the power of ATP binding and hydrolysis to move substances across cellular membranes. Here, we use time-resolved small-angle X-ray scattering combined with conformation-specific nanobodies to directly capture and assign different states to the heterodimeric ABC transporter TM287/288 in solution. Our findings advance our understanding of ABC transporter and demonstrate a broadly applicable method for dissecting the different molecular states in membrane proteins on a second-to-minute time scale. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/679171v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b83fbaorg.highwire.dtl.DTLVardef@5fec81org.highwire.dtl.DTLVardef@1a022fdorg.highwire.dtl.DTLVardef@592c92_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 {micro}g/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the up ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

biophysics↗