Structural basis for amino-acid recognition and transmembrane signalling by tandem Per–Arnt–Sim (tandem PAS) chemoreceptor sensory domains

Abstract

Chemotaxis, mediated by methyl-accepting chemotaxis protein (MCP) receptors, plays an important role in the ecology of bacterial populations. This paper presents the first crystallographic analysis of the structure and ligand-induced conformational changes of the periplasmic tandem Per-Arnt-Sim (PAS) sensing domain (PTPSD) of a characterized MCP chemoreceptor. Analysis of the complex of the Campylobacter jejuni Tlp3 PTPSD with isoleucine (a chemoattractant) revealed that the PTPSD is a dimer in the crystal. The two ligand-binding sites are located in the membrane-distal PAS domains on the faces opposite to the dimer interface. Mutagenesis experiments show that the five strongly conserved residues that stabilize the main-chain moiety of isoleucine are essential for binding, suggesting that the mechanism by which this family of chemoreceptors recognizes amino acids is highly conserved. Although the fold and mode of ligand binding of the PTPSD are different from the aspartic acid receptor Tar, the structural analysis suggests that the PTPSDs of amino-acid chemoreceptors are also likely to signal by a piston displacement mechanism. The PTPSD fluctuates between piston (C-terminal helix) `up' and piston `down' states. Binding of an attractant to the distal PAS domain locks it in the closed form, weakening its association with the proximal domain and resulting in the transition of the latter into an open form, concomitant with a downward (towards the membrane) 4 Å piston displacement of the C-terminal helix. In vivo, this movement would generate a transmembrane signal by driving a downward displacement of the transmembrane helix 2 towards the cytoplasm.

The periplasmic sensing domain of <i>Pseudomonas fluorescens </i>chemotactic transducer of amino acids type B (CtaB): Cloning, refolding, purification, crystallization, and X-ray crystallographic analysis

“…We succeeded in producing folded protein and high-quality crystals by following the refolding procedure that we have recently developed (25,26). The purified protein was monomeric in solution, in line with previous studies that showed PTPSDs from other receptors to be also monomeric in solution (16,20,(25)(26)(27). X-ray crystallographic analysis of CtaB PTPSD in complex with various amino acid ligands is expected to provide an explanation of the structural basis behind the broad ligand specificity of this receptor.…”

“…We have previously observed that expression of periplasmic sensory domains of bacterial MCP receptors in E. coli often results in their deposition predominantly in inclusion bodies (16,20,(25)(26)(27). The recombinant ligand sensing domain of P. fluorescens CtaB is another example of a molecule of this type that required extraction from IBs and refolding.…”

“…The crystal structures of two different PTPSDs with specificity to amino acids have been recently reported, providing first insights into the structural basis of their ligand specificity. Analysis of PTPSD of Campylobacter jejuni Tlp3 in complex with isoleucine (PDB code 4xmr) revealed a strongly hydrophobic pocket accommodating the aliphatic side chain of the ligand, consistent with Tlp3's preference for isoleucine and, likely, other branched amino acids such as valine and leucine (16). The crystal structure of PTPSD of V. cholerae Mcp37 has been reported in complex with alanine and serine (PDB codes 3c8c and 5ave (18)).…”

Cloning, purification, crystallization and X-ray crystallographic analysis of the periplasmic sensing domain of <i>Pseudomonas fluorescens</i> chemotactic transducer of amino acids type A (CtaA)

“…Tlp3 recognizes its ligand isoleucine directly, via its membrane-distal PAS domain (22). The structural analysis of Tlp3 and structure-guided sequence alignments revealed that receptors for amino acids, that have a tandem-PAS sensing domain that recognizes the ligand directly, contain a conserved consensus motif DXXX(R/K)CWYXXA (22). We note that CtaA peri contains this motif and is therefore likely to bind at least some of its amino acid ligands directly.…”

“…Recently, we have reported the crystal structure of the periplasmic sensing domain of C. jejuni transducer-like protein 3 (Tlp3) harboring two PAS domains. Tlp3 recognizes its ligand isoleucine directly, via its membrane-distal PAS domain (22). The structural analysis of Tlp3 and structure-guided sequence alignments revealed that receptors for amino acids, that have a tandem-PAS sensing domain that recognizes the ligand directly, contain a conserved consensus motif DXXX(R/K)CWYXXA (22).…”