The extracellular gate shapes the energy profile of an ABC exporter

Abstract

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter’s conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.

An improved fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification

“…The structure of the complex (Fig. 8 , Table 2 ) was solved by molecular replacement using existing TGP and nanobody structures 58 , 59 , 78 as search models. The asymmetric unit contained two TGP-Sb44 heterodimers, which were highly similar (Cα root mean square deviation of 0.29 Å).…”

“…Data were integrated using XDS 85 , scaled and merged using Aimless 86 . The structure was solved by molecular replacement using Phaser 87 with a TGP monomer from PDB 4TZA 58 and the sybody from PDB 6QV1 78 as the searching templates. The model was built with 2F o -F c maps in Coot 88 , and refined using Phenix 89 .…”

Nanobodies—Useful Tools for Allergy Treatment?

“…Non-immune libraries are typically much larger than immune libraries and a ribosome display was suggested for the initial selection round of such large libraries to work with higher concentrations of nanobody variants than in case of phage display (79,80). Synthetic libraries combined with different selection procedures were successfully used to obtain conformationally selective nanobodies against G protein-coupled receptors (78), sybodies against very challenging targets such as the heterodimeric bacterial ABC exporterTM287/288 (81) or the intracellular KDEL receptor (82) to name a few examples from many others.…”

Potential correlation between carbohydrate‐active enzyme family 48 expressed by gut microbiota and the expression of intestinal epithelial AMP‐activated protein kinase β

“…Accumulating evidence suggests that BA can be oxidized and utilized to synthesize ADP and AMP in intestinal cells, and thus allosterically activate AMPK expression levels. Additionally, changes in molecular morphology (dissociation or oxidation) of SCFA molecules when they enter into intestinal cells may be indicative of ATP-dependent events induced by SCFAs (Hutter et al, 2019).…”

“…dicative of ATP-dependent events induced by SCFAs(Hutter et al, 2019).In order to determine the effect of different experimental diets on the physicochemical features of plasma and animal growth, TMAO, TC, TG, LDL-C (low-density lipoprotein) levels, and weight gain were measured. The results with significantly higher values for these indices were observed in mice fed the red meat diet (RM group, p value <.05,Figure 1b), potentially leading to a higher risk of CVDs(Wu, Micha, & Mozaffarian, 2019).…”