Structural Flexibility of Peripheral Loops and Extended C-terminal Domain of Short Length Substrate Binding Protein from Rhodothermus marinus

Abstract

Substrate binding proteins (SBPs) bind to specific ligands in the periplasmic regions of cells and then bind to membrane proteins to participate in transport or signal transduction. Typically, SBPs consist of two α/β domains and recognize the substrate by a flexible hinge region between the two domains. Conversely, the short-length SBPs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes. We previously determined the crystal structure of Rhodothermus marinus SBP (named as RmSBP), consisting of a single α/β domain; however, the substrate recognition mechanism is still unclear. To better understand the functions of short length RmSBP, we performed a comprehensive study, involving comparative structure analysis, computational substrate docking, and X-ray crystallographic data. RmSBP shares a high level of similarity in the α/β domain region with other SBPs, but it has a distinct topology in the C-terminal domain. The substrate binding model suggested that conformational changes in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility in the β1–α2 loop, β5–β6 loop, and extended C-terminal domains, based on the electron density map and temperature B-factor analysis. These results provide information that will further our understanding on the functions of the short length SBP. Supplementary Information The online version contains supplementary material available at 10.1007/s10930-021-09970-z.

B-Factor Rescaling for Protein Crystal Structure Analyses

“…Sánchez Rodríguez and colleagues examined the importance of B-factors in molecular replacement computations for solving the phase problem in protein X-ray crystallography [14]. Bae and colleagues analyzed the structural flexibility in two loops and in the C-terminal region of Rhodothermus marinus substrate binding protein to reach a deeper understanding of the binding mechanism [15]. Johnson and coworkers related B-factors to drug potency-rizotinib and lorlatinib-against C-ros oncogene 1 kinase and anaplastic lymphoma kinase [16].…”

Structural and bioinformatics analysis of single-domain substrate-binding protein from Rhodothermus marinus

“…Crystallographic analysis of Rhodothermus marinus SBPs (RmSBPs) showed that they had structural similarity to the C-terminal α/β domains of l -tryptophan-bound SBP from Streptococcus pneumonia (SpSBP) and phenylalanine-bound SBP from Vibrio cholerae ) O1 biovar El Tor str. N16961 (VcSBP) but did not contain the N-terminal α/β domains [ [11] , [12] , [13] ]. The surface of the amino acid-binding position of SpSBP and VcSBP differed from the structurally identical position of RmSBP [ 11 ].…”

“…The surface of the amino acid-binding position of SpSBP and VcSBP differed from the structurally identical position of RmSBP [ 11 ]. Computational analysis of the putative ligand binding sites of RmSBP indicated that these had identical structure positioning as the amino acid-binding site of SpSBP and VcSBP [ 12 ]. The putative substrate-binding site of RmSBP had a rigid structural folding while the β1–α2 and β5–β6 loops and extended C-terminal domains showed relatively high molecular flexibility [ 12 ].…”

“…Computational analysis of the putative ligand binding sites of RmSBP indicated that these had identical structure positioning as the amino acid-binding site of SpSBP and VcSBP [ 12 ]. The putative substrate-binding site of RmSBP had a rigid structural folding while the β1–α2 and β5–β6 loops and extended C-terminal domains showed relatively high molecular flexibility [ 12 ]. On the other hand, the amino acid sequence of RmSBP shows a 20% similarity with that of ORF1ab polyprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain HKU-SZ-001_2020 [ 14 ].…”

Data of radiation damage on selenomethionine-substituted single-domain substrate-binding protein

“…Typical SBP proteins recognize a substrate using two α/β domains [12] , whereas the single-domain substrate-binding protein from Rhodothermus marinus (RmSBP) has a single α/β domain. The crystal structure and flexibility of RmSBP have been established [12] , [13] , [14] , but its molecular function is unknown. To better understand this process, radiation damage to selenomethionine-substituted RmSBP (SeMet-RmSBP) was used as a model sample, rather than focusing on the identification of biological function [1] .…”