Identification of additional mechanistically important residues in the multidrug transporter styMdtM of Salmonella...

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Multidrug efflux is a critical resistance mechanism in bacterial pathogens like Salmonella Typhi.
• The multidrug efflux transporter styMdtM (STY4874) from the major facilitator superfamily is expressed in S. Typhi.
• Previous functional assays highlighted residues crucial for styMdtM transport activity.

Purpose of the Study:

• To identify additional residues influencing styMdtM transport activity using computational modeling and mutagenesis.
• To elucidate the structural and functional roles of specific amino acid residues in the transporter.
• To advance the mechanistic understanding of this clinically relevant multidrug transporter.

Main Methods:

• Utilized an AlphaFold model to predict residue importance for mutagenesis.
• Performed site-directed mutagenesis on identified residues (Cys185, Tyr29, Tyr231, Gln294).
• Conducted functional transport assays and X-ray crystallography for structural analysis.

Main Results:

• Mutation of peripheral Cys185 did not affect transporter structure or function.
• Substitution of channel-lining Tyr29 and Tyr231 completely abolished transport activity.
• Mutation of Gln294, facing peripheral helices, partially impaired transport of certain substrates.
• Wild-type styMdtM crystallization yielded diffraction data (4.5 Å resolution) with established unit cell parameters (a=b=64.3 Å, c=245.4 Å, α=β=γ=90°, space group P4).

Conclusions:

• Identified critical channel-lining residues (Tyr29, Tyr231) essential for styMdtM function.
• Demonstrated that peripheral residues can have varying impacts on transporter activity.
• Provided structural and functional data contributing to a mechanistic understanding of styMdtM.
• These findings represent a step towards understanding multidrug resistance in S. Typhi.