RESUMO
We propose that the alternative crystal forms of outward open UlaA (which are experimental, not simulated, and contain the substrate in the cavity) can be used to interpret/validate the MD results from MalT (the substrate capture step, which involves the mobile second TMSs of the V-motifs, TMSs 2 and 7). Since the crystal contacts are the same between the two alternative crystal forms of outward open UlaA, the striking biological differences noted, including rearranged hydrogen bonds and salt bridge coordination, are not attributable to crystal packing differences. Using transport assays, we identified G58 and G286 as essential for normal vitamin C transport, but the comparison of alternative crystal forms revealed that these residues to unhinge TMS movements from substrate-binding side chains, rendering the mid-TMS regions of homologous TMSs 2 and 7 relatively immobile. While the TMS that is involved in substrate binding in MalT is part of the homologous bundle that holds the two separate halves of the transport assembly (two proteins) together, an unequal effect of the two knockouts was observed for UlaA where both V-motifs are free from such dimer interface interactions.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Conformação Proteica , Ácido Ascórbico/química , Cristalografia por Raios X , Dimerização , Proteínas de Escherichia coli/ultraestrutura , Ligação de Hidrogênio , Proteínas de Membrana Transportadoras/ultraestrutura , Especificidade por SubstratoRESUMO
The Δ-distance maps can detect local remodeling that is difficult to accurately determine using superimpositions. Transmembrane segments (TMSs) 11 in both LacY and XylE of the major facilitator superfamily uniquely contribute the greatest amount of mobile surface area in the outward-occluded state and undergo analogous movements. The intracellular part of TMS11 moves away from the C-terminal domain and into the substrate cavity during the conformational change from the outward-occluded to the inward-occluded state. A difference was noted between LacY and XylE when they assumed the inward open state after releasing a substrate to the inside in which TMS11 of LacY moved further into the substrate release space, whereas in XylE, TMS11 slightly retracted into the C-terminal domain. Independent movement of the N-terminal half of TMS11 suggests that it is flexible in the middle. Repeat-swapped homology modeling was used to discover that a loop connecting TMSs 10 and 11 in LacY probably moves during the transition between the unavailable outward-open state and the outward-occluded state. TMSs 11 and the other elements displaying a notable domain-independent movement colocalize with the interdomain linker, suggesting that these elements could drive the alternating access movement between the domain halves. Preliminary evidence indicates that analogous movements occur in other members of the major facilitator superfamily.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Simportadores/química , Simportadores/metabolismo , Conformação Proteica , Homologia Estrutural de ProteínaRESUMO
We introduce the value of information obtained by comparing alternative crystal forms of the same sub-state (of outward open UlaA, our example protein), which is found in the same lattice configuration but different space groups. We compare instability estimates obtained using this new method (alternative crystal forms) with temperature factors. Using a transport assay result, we correlate observations for two homologous secondary structure elements, and show that the alternative states method for obtaining instability estimates provide differentiating information about an important and immobilized mid-TMS region. The data presented in this article are related to the article entitled "The V-motifs facilitate the substrate capturing step of the PTS elevator mechanism" (A. Vastermark, A. Driker, J. Weng, X. Li, J. Wang, M.H. Saier Jr., 2016).