Pyridoxamine is a substrate of the energy-coupling factor transporter HmpT

Abstract

Energy-coupling factor (ECF) transporters belong to a novel family of proteins that forms a subset within the ATP-binding cassette (ABC) transporter family. These proteins are responsible for the uptake of micronutrients in bacteria. ECF transporters are composed of four proteins: the A- and A'-components, the T-component and the S-component. One of the ECF transporters, named HmpT, was crystallized in the apo form with all four components. It is currently unknown whether HmpT serves as a transporter for hydroxymethyl pyrimidine or the different forms of vitamin B6 (pyridoxine, pyridoxal or pyridoxamine). Using a combination of molecular dynamics (MD) simulations and mass spectrometry, we have identified pyridoxamine to be the preferred substrate of HmpT. Mass spectra show that the mass of the substrate from the HmpT-substrate complex matches that of pyridoxamine. MD simulations likewise indicate that pyridoxamine interacts most strongly with most of the conserved residues of the S-component (Glu 41, His 84 and Gln 43) compared with the other vitamin B6 forms. Furthermore, the simulations have implied that loops 1 and 5 of the S-component can participate in the gating action for HmpT.

Keywords: ECF transporters; HmpT; S-component; gating mechanism; mass spectrometry; molecular dynamics.

Figure 1

Cartoon representation of the ECF transporter HmpT. The regions in blue and red are the A-components while the S- and T-components are colored orange and green, respectively.

Figure 2

Structure of the S-component, HmpT and substrates used in the simulation. (a) Features a snapshot of pyridoxamine inside the binding pocket of the S-component, HmpT (4HZU, [11]), that is being simulated in the open orientation. In this snapshot, loop 1 is starting to close over the opening of the binding pocket. (b) Displays the structures of the ligands that were simulated with HmpT: pyridoxine (pdx), pyridoxal (pdxl), pyridoxamine (pdxm) and hydroxymethyl pyrimidine (hmp). The white, cyan, blue and red balls represent hydrogen, carbon, nitrogen and oxygen atoms, respectively.

Figure 3

Mass spectra of the standard and the substrate extracted from EcfS protein. (a, b) Show the pyridoxamine dihydrochloride solution (CAS 524-36-7, SIGMA-ALDRICH P9380). (c, d) Show the supernatant sample extract from HmpT protein precipitate by methanol/water (1:1 v/v) and sonication treatment. All the samples were analyzed by Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with UPLC. The MS spectra and MS/MS spectra were acquired with resolution of 70 000 and 17 500, respectively.

Figure 4

Snapshots of interactions between pyridoxamine and selected residues in the binding pocket. (a) Represents close-up snapshots of pyridoxamine interacting with the conserved residues Glu 41, Tyr 120 and Gln 143. Similar snapshots of pyridoxamine interacting with Trp 76 and Ser 136 are displayed in (b).

Figure 5

The closing of the gate in the presence of ligand as seen through the opening of the S-component. The S-component is drawn in surface rendering (a) and in cartoon rendering (b). The region colored orange in (a) represents Loop 1. The right side of (b) shows loop 1 closing over the opening of the binding pocket.

Figure 6

Total distance of the center of mass of pyridoxamine to the centers of mass of the side chains of the conserved residues. (a) Represents data from the simulation of HmpT in the closed conformation, while (b) represents data from the open conformation. The labels inside the boxes show the type of mutation that was imposed on HmpT.