Experimental and computational mapping reveals preferential nanobody targeting of the intracellular cavity of the amino acid transporter BasC

Nanobodies are valuable tools for stabilizing and investigating membrane transporters, but the systematic characterization of large nanobody collections remains experimentally demanding. Here, we combined biochemical and transport assays, experimental structural data and AlphaFold3 modelling to characterize a repertoire of 29 nanobodies targeting the bacterial amino acid transporter BasC. By exploiting the random orientation and asymmetric apparent substrate affinities of BasC in proteoliposomes, comparison of nanobody-mediated influx and efflux inhibition enabled experimental assignment of binding sidedness. The collection showed a marked bias towards inhibition from the intracellular face, whereas only two, Nb58 and Nb78, were identified as extracellular binders. Compatibility assays and a 6.1 [A] crystallographic map of the BasC-Nb53-Nb58 complex supported the simultaneous binding of Nb53 and Nb58 to opposite sides of the transporter and validated the functional sidedness assignment. AlphaFold3 generated high-confidence interaction models for 16 of the 29 complexes. Ten of these models targeted the large intracellular cavity, revealing recurrent epitope clusters. The predicted binding modes of Nb53, Nb71 and Nb74 agreed with available experimental structures, supporting the accuracy of these predictions. Notably, these three nanobodies had previously provided the most informative structural and smFRET data. Our results show that transport-based sidedness measurements together with confidence-filtered structural predictions can define the functional and structural landscape of a nanobody repertoire. This integrated approach may provide a virtual structural screening strategy to prioritize nanobodies, identify dominant epitope regions and select potentially compatible combinations for experimental and structural studies.