... understanding life in molecular detail

Prof Adrian Goldman

Structural studies of Membrane-integral and associated systems


My research focuses on understanding interactions in or near the membrane.  We are interested in bacterial autotransporter secretion and the host-pathogen interactions that lead to disease; we are interested in cell signalling in the RET tyrosine kinase receptor system; and we are interested in integral membrane pumps and channels, such as the T. maritima pyrophosphatase that we solved in 2012.

Current major projects include:
  • Structure-funtion studies of integral membrane pyrophosphatases
  • Understanding how trimeric autotransporters work
  • Bacterial evasion of complement
  • The mechanism of the RET tyrosine kinase

Our goal is to understand the structure and function of various biological systems at the atomic level, in particular membrane-integral and membrane- associated proteins. We wish to understand how they work – whether in transmitting signals, in binding to other proteins, or in pumping protons to conserve energy. We primarily use x-ray crystallography, supplemented with functional, mutagenesis and theoretical studies.  One example of our work is on the structure of membrane-integral pyrophosphatases.

Plants, parasitic protozoa, bacteria and archae contain membrane-integral pyrophosphatases (M-PPases). These are novel primary transmembrane pumps with 14-17 transmembrane (TM) helices that link pyrophosphate (PPi) hydrolysis to sodium or proton pumping (1). PPases are essential to drive anabolic reactions like DNA synthesis to completion. Unlike the soluble PPases, M-PPases recycle part of the free energy of PPi hydrolysis to generate electrochemical potential across biological membranes.  In plants, they are vital for maturation and enhance survival under abiotic stress conditions (drought, anoxia, cold) (2). They also are important for proliferation of disease causing protozoa (3).

M-PPases have neither sequence, structural nor functional similarity to the FoF1 or P-type ATPases. They can either be proton or sodium pumps, and some require K+ for maximal activity. The resting enzyme state is EMg2, and two more metal ions bind with substrate, Mg2PP i (2).

We have solved the structure of the Na+-pumping M-PPase of Thermotoga maritima (TmPPase) in the resting state (TmPPase:Ca:Mg) at 2.6 Å resolution and with product bound (TmPPase:Pi2:Mg4) at 4.0 Å. TmPPase is a homodimer with 16 TM-helices per monomer (Fig. 1). The helices extend up to 25 Å from the membrane bilayer on the cytoplasmic side so that the hydrolytic centre is held some 20 Å above the membrane plane.

The protein has a completely novel fold. The unusual active site has four distinct regions: the hydrolytic centre, a “coupling funnel”, the gate (closed in our structure) just below the membrane surface, and an exit channel for Na+-ions.  The distance from the hydrolytic centre to the gate is about 20 Å. Six helices (5-6, 11-12 and 15-16) form the hydrolytic centre and the coupling funnel on the cytoplasmic face of the protein.  However, just four helices (5, 6, 12 and 16) form the gate and channel, creating an internal “symmetry mismatch” between the two sides of the protein.

Below the hydrolytic centre is an unusual “coupling funnel” (Fig. 1) containing eight absolutely conserved charged residues that couple hydrolysis to ion pumping. Some of the interactions there in the resting structure suggest that the funnel is poised to switch into an alternate conformation to pump the sodium ion, especially R191, which is almost equidistant between D243 and D236. Alignment of our structures and that of the substrate complex of the related mung bean PPase (4) shows that TM12 moves downwards by about 2 Å, concomitant with R191 switching up and down in a locked and released spring movement.

As the changes in the inhibited and product-bound states are similar, with the exit channel closed in both, we suggest that the ion pumping in M-PPases occurs via a “binding change” mechanism as in the FoF1 ATP synthase, and that the full conformational change that opens the gate involves a downwards piston-like motion of TM12 (Fig 2).  This is driven by a concerted contraction during substrate binding. This change destroys a high-affinity sodium-binding site above the gate, leading to transfer of the ion into the exit channel, followed by its rapid diffusion into the periplasm. Consistent with this model, there are no conserved polar residues below the gate. Release of the sodium ion would allow the gate to close, hydrolysis to occur, product to leave, and the protein to revert to its resting state.

Our work and the related mung bean structure provide the first steps in understand structure and mechanism in this novel family of ion pumps.

A B
C

 

Figure 1: Structure of membrane bound Thermotoga maritima membrane pyrophosphatase (Tm-PPase) and comparison of different catalytic states. A) Dimer of Tm-PPase in relation to the lipid bilayer: one active site site is indicated by the green metal ion (Mg2+). B) Comparison of the apo-structure (light blue) and Pi-product complex (active site view form top) of Tm-PPase (green), showing the contraction of the structure around the active site with bound ligand (2 x Pi, shown as spheres). C) Comparison of the Tm-PPase apo-structure (light blue), product complex (green), and mung bean PPase inhibitor complex (brown) around the gate region, showing the ionic bonds and movement of TM-helix 12 (A and C, adapted from Kellosalo et al. 2012)

References:

1. Malinen, AM, Belogurov, GA, Baykov, AA, Lahti, R, Biochemistry, 46:8872–8878(2007).

2. Maeshima, M, Biochim. Biophys. Acta, 1465:37–51(2000).

3. McIntosh, MT, Vaidya, AB, Int. J. Parasitol., 32:1–14(2002).

4. Lin, SM, Tsai, JY, Hsiao, CD, Huang, YT, Chiu, CL, Liu, MH, Tung, JY, Liu, TH, Pan, RL, Sun, YJ, Nature, 484:399–403(2012).

 

 

 

Detailed research programme                  Close ▲
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Leadership Chair in Membrane Biology, Leeds, present
PhD
Wolfson Award, Royal Society 2013-2018, Lucille P. Markey Charitable Trust Research Fellow 7.1987-12-1991, Johnson & Johnson Discovery Research Fellow 4.89 â?? 4.91, Henry Rutgers Fellow 7.88 â?? 7.91

Professor (University of Turku) (1997 - 1999)
Head of Research (University of Helsinki) (1999-2007)
Director of Research (University of Helsinki), (2008-2013)
Professor (University of Helsinki) (2013)

Astbury 6.108b
School of Biomedical Sciences
0113 343 8537
a.goldman@leeds.ac.uk

Selected Publications

  1. Kellosalo, J., Kajander, T., Kogan, K., Pokharel, K. & Goldman, A. The Structure and Catalytic Cycle of a Sodium-Pumping Pyrophosphatase. Science 337, 473-476 (2012).

  2. Kajander, T., Lehtinen, M. J., Hyvärinen, S., Bhattacharjee, A., Leung, E., Isenman, D. E., Meri, S., Goldman, A. & Jokiranta, S. T. Structure of the FH19-20:C3d complex explains both target recognition and aHUS pathogenesis. Proc. Natl. Acad. Sci U.S.A., 108, 2897-2902 (2011).

  3. Leo, J. C., Lyskowski, A., Hattula, K., Hartmann, M., Schwarz, H., Butcher, S. J., Linke, D., Lupas, A. N. & Goldman, A. The structure of E. coli IgG-binding protein D suggests a general model for bending and binding in trimeric autotransporter adhesins. Structure 19, 1021–1030 (2011).

  4. Nummelin, H., Merckel, M. C., Leo, J., Lankinen, H. Skurnik, M. & Goldman, A.  The structure of Yersinia adhesin YadA collagen-binding domain is a novel left-handed parallel beta-roll. EMBO J., 23, 701-711 (2004).