Substrate-binding proteins (SBPs), selectively capture ligand(s) and ensure their translocation via its cognate ATP-binding cassette (ABC) import system. SBPs bind their cognate ligand(s) via an induced-fit mechanism known as the “Venus Fly-trap”; however, this mechanism lacks the atomic details of all conformational landscape as the confirmatory evidence(s) in its support. In this study, we delineate the atomic details of an SBP, β-glucosides-binding protein (βGlyBP) from Thermus thermophilus HB8. The protein βGlyBP is multi-specific and binds to different types of β-glucosides varying in their glycosidic linkages viz. β-1,2; β-1,3; β-1,4 and β-1,6 with a degree of polymerization of 2–5 glucosyl units. Structurally, the protein βGlyBP possesses four subdomains (N1, N2, C1 and C2). The unliganded protein βGlyBP remains in an open state, which closes upon binding to sophorose (SOP2), laminari-oligosaccharides (LAMn), cello-oligosaccharides (CELn), and gentiobiose (GEN2). This study reports, for the first time, four different structural states (open-unliganded, partial-open-unliganded, open-liganded and closed-liganded) of the protein βGlyBP, revealing its conformational changes upon ligand binding and suggesting a two-step induced-fit mechanism. Further, results suggest that the conformational changes of N1 and C1 subdomains drive the ligand binding, unlike that of the whole N- and C-terminal domains (NTD and CTD) as known in the “Venus Fly-trap” mechanism. Additionally, profiling of stereo-selection mechanism for α- and β-glucosides reveals that in the ligand-binding site four secondary structural elements (L1, H1, H2 and H3) drive the ligand selection. In summary, results demonstrate that the details of conformational changes and ligand selection are pre-encoded in the SBPs.

底物结合蛋白（SBP）选择性捕获配体，并通过其关联的ATP结合盒（ABC）导入系统确保其易位。SBP通过称为“维纳斯捕蝇器”的诱导拟合机制结合其同源配体；然而，该机制缺乏所有构象景观的原子细节作为其支持的确认证据。在这项研究中，我们描述了嗜热栖热菌的SBP，β-糖苷结合蛋白（βGlyBP）的原子细节HB8。βGlyBP蛋白是多特异性的，可与糖苷键不同的不同类型的β-糖苷结合。β-1,2; β-1,3; β-1,4和β-1,6的聚合度为2–5个葡萄糖基单元。βGlyBP蛋白在结构上具有四个亚结构域（N1，N2，C1和C2）。未配体的蛋白质βGlyBP保持开放状态，当与槐糖（SOP2），板层低聚糖（LAMn），纤维寡糖（CELn）和龙胆二糖（GEN2）结合时即关闭。这项研究首次报道了蛋白质βGlyBP的四个不同结构状态（开放-未配体，部分开放-未配体，开放-配体和封闭-配体），揭示了其与配体结合后的构象变化，并提出了两个步骤诱导拟合机制。进一步，结果表明，N1和C1子域的构象变化驱动配体结合，这与“维纳斯捕蝇器”机制中已知的整个N和C端域（NTD和CTD）不同。另外，对α-和β-葡萄糖苷的立体选择机制的分析揭示了在配体结合位点，四个次级结构元件（L1，H1，H2和H3）驱动配体选择。总而言之，结果表明构象变化和配体选择的详细信息已在SBP中预先编码。