Arg302 governs the pK _a of Glu325 in LacY

Abstract

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H+, using the free energy released from downhill translocation of H+ to drive accumulation of galactosides against a concentration gradient. In neutrophilic Escherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pKapp) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pKa is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pKa. The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.

The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY

“…Most recently, side chains in the vicinity of Glu325 were mutated with the rationale that interaction with Glu325 should alter its pK a . Remarkably, only mutant R302K of the sites tested causes the pK a to decrease and by over 2 pH units, thereby lending further support to the idea that H + is displaced from Glu325 by spatial fluctuations in the position of Arg302 moving near Glu325 or vice versa (40). The possibility will be tested with a suppressor t-RNA approach using arginine homologs with side chains of varied lengths (43).…”

“…close to Glu325 (∼6 Å), are unable to catalyze active transport but exhibit transmembrane exchange (38), and evidence has been presented (39,40) in support of the idea that H + may be extracted from Glu325 by spatial fluctuations in the position of Arg302 that expel H + by moving near Glu325 or vice versa.…”

Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily

“…Compatible with this flexible design of membrane-embedded substrate-and H + -binding sites, neither D34 in MdfA nor the drug/H + stoichiometry is conserved within the DHA1 subfamily 51 . By contrast, in the substrate-specific LacY, the substrate/ H + coupling relies on the extensive and intricate H-bonding networks, as such, the precise positioning of H + -and substrate-binding amino acids is essential for the transport function 11,46 . Therefore, the substrate/H + coupling mechanism of LacY lacks flexibility and the substrate/H + stoichiometry is fixed.…”

“…Moreover, the coupling between substrate-and H + -binding in the H + -dependent LacY is highly specific, which depends on the dense and precise networks of H-bonds implicated in substrate-and H + -binding. Therefore, the substrate/H + coupling in LacY is intolerant to the alteration of amino acids involved in these H-bonding interactions 10,11 . Apparently, the exact chemical structure of the LacY substrate imposes stringent structural restraints on the substrate/H + coupling mechanism 8 .…”

Monoclonal antibody 4B1 influences the pKa of Glu325 in lactose permease (LacY) from Escherichia coli: evidence from SEIRAS

“…In neutrophilic E. coli , the internal pH is kept at ~ 7.6 over the physiological range, but the apparent p K (p K app ) for galactoside binding is 10.5 [5]. Surface‐enhanced infrared absorption spectroscopy (SEIRAS) demonstrated that the high p K a is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively [6,10]. The microenvironment of this residue is shown in Fig.…”