Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Structure of a dimeric tripartite ATP-independent periplasmic (TRAP) transporter from Haemophilus influenzae. (#213)

Michael Currie 1 , James Davies 2 , Mariafrancesca Scalise 3 , Michael Griffin 4 , David Drew 2 , Cesare Indiveri 3 , Rachel North 2 , Renwick Dobson 1
  1. School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
  2. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
  3. Department DiBEST (Biologia, Ecologia, Scienze della Terra) , University of Calabria, Calabria, Italy
  4. Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Australia

Tripartite ATP-independent periplasmic (TRAP) transporters are a family of secondary transporters that import organic acids, such as sialic acid, into bacterial and archaeal cells. TRAP transporter systems comprise three components––a soluble substrate-binding protein that binds the substrate in the periplasm and two transmembrane subunits that then move the substrate across the membrane. In ~25% of predicted TRAP systems, including the Haemophilus influenzae SiaQM TRAP system, the two membrane components are fused. We set out to determine the oligomeric state and high-resolution molecular structure of the fused HiSiaQM TRAP to understand its mechanism of action.

We first studied the behaviour of the integral membrane components (HiSiaQM) in various detergents and amphipol using biophysical methods revealing that in all cases the protein exists in a monomer-dimer self-association. We assayed the activity of L-MNG solubilised HiSiaPQM reconstituted into proteoliposomes and find that it is comparably active to previous studies that solubilised HiSiaPQM in DM, including requiring both a Na+ gradient and the substrate-binding protein (HiSiaP) for activity. We next determined the nature of the interaction between HiSiaP and HiSiaQM using analytical ultracentrifugation, which demonstrates that the KD for full complex formation (HiSiaPQM) in the presence of sialic acid is in the micromolar range.

Lastly, we determined the structure of the integral membrane components (HiSiaQM) to 2.96 Å using single-particle cryo-electron microscopy. The structure reveals how fused TRAP systems use extra scaffolding transmembrane and amphipathic helices to connect the two membrane bound subunits. Remarkably, the transporter forms two distinct populations of stable dimers when solubilised in amphipol. Side-by-side and inverted orientations were determined to 3.26 Å and 2.96 Å with each protomer either facing the same way or with one protomer rotated 180° relative to the other. This work supports our proposed ‘elevator-with-an-operator’ mechanism of membrane transport by TRAP transporters and provides a framework for antimicrobial design, blocking the SiaPQM interaction or sialic acid binding site and inhibiting bacteria that rely solely on SiaPQM for obtaining sialic acid, such as H. influenzae.