Biomolecular Interaction Centre, Maurice Wilkins Centre for Biodiscovery, MacDiarmid Institute for Advanced Materials and Nanotechnology, and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
Biomolecular Interaction Centre, Maurice Wilkins Centre for Biodiscovery, MacDiarmid Institute for Advanced Materials and Nanotechnology, and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
Mariafrancesca Scalise
Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy
Ashutosh Gulati
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
Biomolecular Interaction Centre, Maurice Wilkins Centre for Biodiscovery, MacDiarmid Institute for Advanced Materials and Nanotechnology, and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
Michael C Newton-Vesty
Biomolecular Interaction Centre, Maurice Wilkins Centre for Biodiscovery, MacDiarmid Institute for Advanced Materials and Nanotechnology, and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
Gayan S Abeysekera
Biomolecular Interaction Centre, Maurice Wilkins Centre for Biodiscovery, MacDiarmid Institute for Advanced Materials and Nanotechnology, and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
Ramaswamy Subramanian
Biological Sciences and Biomedical Engineering, Bindley Bioscience Center, Purdue University West Lafayette, West Lafayette, United States
Department of Chemistry and Molecular Biology, Biochemistry and Structural Biology, University of Gothenburg, Gothenburg, Sweden
Rosmarie Friemann
Centre for Antibiotic Resistance Research (CARe) at University of Gothenburg, Gothenburg, Sweden
Jane R Allison
Biomolecular Interaction Centre, Digital Life Institute, Maurice Wilkins Centre for Molecular Biodiscovery, and School of Biological Sciences, University of Auckland, Auckland, New Zealand
Biochemistry Department, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
Michael DW Griffin
ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio Molecular Science and Biotechnology Institute, Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Australia
Borries Demeler
Department of Chemistry and Biochemistry, University of Montana, Missoula, United States; Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
Soichi Wakatsuki
Biological Sciences Division, SLAC National Accelerator Laboratory, Menlo Park, United States; Department of Structural Biology, Stanford University School of Medicine, Stanford, United States
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden
Cesare Indiveri
Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy; CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Bari, Italy
Biomolecular Interaction Centre, Maurice Wilkins Centre for Biodiscovery, MacDiarmid Institute for Advanced Materials and Nanotechnology, and School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio Molecular Science and Biotechnology Institute, Department of Biochemistry and Pharmacology, University of Melbourne, Melbourne, Australia
Rachel A North
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden; School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, Australia
Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.
