Substrate-binding proteins (SBPs) bind to specific ligands and are associated with membrane protein complexes for transport or signal transduction. Most SBPs recognize substrates by the hinge motion between two distinct alpha/beta domains. However, short SBP motifs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes, but structural and functional studies have yet to be performed. Here, we report the crystal structure of an unusually small SBP from Rhodothermus marinus (named as RmSBP) at 1.9 angstrom. This protein is composed of a single alpha/beta-domain, unlike general SBPs that have two distinct domains. RmSBP exhibits a high structural similarity to the C-terminal domain of the previously reported amino acid bound SBPs, while it does not contain an N-terminal domain for substrate recognition. As a result of the structural comparison analysis, RmSBP has a putative SBP that is different from the previously reported SBP. Our results provide insight into a new class of substrate recognition mechanism by the mini SBP protein. (C) 2018 Elsevier Inc. All rights reserved.