Visualizing the Structure and Function of Integral Membrane Proteins C. Watters1, D. Guertin2, D. Conache2, and D. Koparov2 Department of Biology1 and Library and Informational Services2, Middlebury College 5 July 2005

Watters, et al., 2005, Middlebury College Introduction & Goals Create dynamic models of integral membrane proteins (IMP). Integrate functional and structural aspects of IMP simply but accurately. Render IMP behavior in a stochastic manner. Several Goals: Especially important to show students IMP behavior in n a kinetic context involving random molecular and ionic movement. Watters, et al., 2005, Middlebury College

The Na+/K+ Pump: A Case in Point As a beginning, took possibly the most commonly discussed membrane protein, the “pump” responsible for maintaining the asymmetric gradients of Na and K across the cell’s outer membrane. Note two sub-units: alpha and beta, with alpha responsible for harnessing the energy released from the hydrolysis of ATP to transport Na outwards and K inwards. Thus, want to model the linkage of the pump’s catalytic and transport functions. Those two functions best illustrated by the Post-Albers model of the Pump Watters, et al., 2005, Middlebury College

The Post-Albers Model of the Na+/K+ Pump Enzymatic Model for ATP hydrolysis linked with activation/transport of Na and K; Therefore cyclical. ATP hydrolysis occurs in (at least) 2 steps, with transiently phosphorylated enzyme intermediate (at least) 2 transient states of the enzyme - E1 and E2 – and 2 conformational changes between these states E1 is Na-sticky form is open to cytoplasm; becomes phosphorylated E2 is “K-sticky” form open to outside; rapidly dephosphorylates. This pumping operation illustrated less abstractly in text book cartoons, e.g., Watters, et al., 2005, Middlebury College

Pump Structure and Function: A Static Cartoon Most text books illustrate pump structure/function according to Albers-Post model in this fashion. This is the alpha subunit. Note location of ion binding and phosphorylation sites on large cytoplasmic domain. Note two conformational changes accompanying transport and hydrolysis. We wanted to present a more dynamic and, as it turned out, more accurate model of this process as far as ATP hydrolysis is concerned BUT: High resolution crystallographic data is not yet available for the Na/K pump…BUT it is for the Ca-pump and the structures are very similar, as evidenced in the next slide. Watters, et al., 2005, Middlebury College

The Calcium Pump E1 State E2 State Note changes in orientation of 3 cytoplasmic domains and the effect of these changes on the organization of the transmembrane domains. Note bound Ca in E1 state (analogous to Na?) and its release from the E2 state as helices change their binding affinities and open to the exterior (“up” in Figure). Let’s examine the static features of these two states in more detail. (from PDB, after Toyoshima, et al., 2000; Xu, et al., 2002) Watters, et al., 2005, Middlebury College

Conservation of Pump Structures Na+/K+ Pump Ca2+ Pump This Figure illustrates the primary structure of two pump proteins: the Na/K pump and the Ca-pump. Note similar transmembrane organization of 10 transmembrane domains: primary structure not highly conserved in these regions. Cytoplasmic domains attached to transmembrane helices 1, 2 & 3, and 4 & 5 and are very highly conserved, however. These consist of a nucleotide-binding domain (N), a domain containing the Phosphorylation motif (P), and an Activation domain (A). These 3 domains are the catalytic center of the pump. They cooperate to hydrolyze ATP and effect the changes in the 10 transmembrane helices necessary for selective transport: - changes in binding affinities for Na and K - changes in helix “open” orientation. We decided to model the catalytic activity of the Na/K pump using the well recently well-characterized structure of the Ca-pump catalytic domains. Watters, et al., 2005, Middlebury College

Watters, et al., 2005, Middlebury College E1 Structure Helical membrane domains (M) open to cytoplasm; “Na+ sticky”. Nucleotide- (N) and Phosphate- (P) binding domains separated from Activation (A) domain. The N domain is green, the P domain is yellow and the A domain is blue. Transmembrane helices attached to these domains have similar colors. ATP bound to N domain. Watters, et al., 2005, Middlebury College

Watters, et al., 2005, Middlebury College E2 Structure Helical membrane domains open to exterior; “K+ sticky”. ATP hydrolyzed: ADP released; P domain phosphorylated. P, N, A domains associated, forming active catalytic unit. Note especially how the transmembrane domains change their organization with the change in orientation of A, P and N. Watters, et al., 2005, Middlebury College

Creating A Domain Structure Drawing splines and “skinning” the ATP-binding domain (N) using LightWave 3D. Protein Explorer was used to create the usual tertiary structure for the three catalytic domains, here exemplified by N. The Nucleotide-binding motif is colored yellow. Major and minor splines are drawn and the resulting structure is covered with a “skin”, with a pocket created for the N-motif. Similar models were created for the P and A domains. The transmembrane helices were represented by cylinders. Watters, et al., 2005, Middlebury College

Assembled ATPase of the Na+/K+ Pump Watters, et al., 2005, Middlebury College

Na+/K+ Pump (a-subunit) E1 E2 The E1 and E2 states were drawn to illustrate the relative locations of the catalytic domains. Helices 8, 9 and 10 were cut away to illustrate the interior of the pump. The beta subunit was also removed. The relative orientation and binding affinities of the transmembrane domains were more abstractly drawn. Watters, et al., 2005, Middlebury College

Placing the Pump in a Membrane Perspective The Heart of LightWave 3 D, which creates a perspective (note camera location) and allows for motion interpolation among the various models as the catalytic cyclic proceeds. Watters, et al., 2005, Middlebury College

The Na+/K+ Pump. THE MOVIES Pumping Ions 1 PI 2. Ouabain’s Revenge: the Pump Halted PI 3. Pump Redux, or Overwhelmed by the Gradients. PI 4. The Great Na+ - Na+ Exchange PI 5. The Great K+ - K+ Exchange Watters, et al., 2005, Middlebury College

Limitations of Pump Models Crystallographic structures may not be accurate “native” structures. Organization of transmembrane helical domain is inaccurate. Helices not parallel; no “channel” evident. Binding sites may not be arranged as depicted. Na+- and K+-binding sites may be different: likely overlapping. Cooperative effects of N, P and A domains (= catalysis) on transmembrane domain likely more complex than shown. Watters, et al., 2005, Middlebury College

Watters, et al., 2005, Middlebury College Other Integral Membrane Proteins: Tyrosine Kinase and G-protein-linked Hormone Receptors Watters, et al., 2005, Middlebury College

Tyrosine Kinase Receptors Initial Events: EGF Binding to EGFR Watters, et al., 2005, Middlebury College

Tyrosine Kinase Receptors Downstream Transduction Events Watters, et al., 2005, Middlebury College

Tyrosine Kinase Receptors Kinase Inhibition by 4-Anilinolquinazoline David –put PDB 1M17 Here, to same Scale, highlighting inhibitor. EGFR - Kinase Domain Watters, et al., 2005, Middlebury College

Watters, et al., 2005, Middlebury College Other Integral Membrane Proteins: Signaling by Tyrosine Kinase and G-protein-linked Hormone Receptors David: provide link from each still to a QT movie. Watters, et al., 2005, Middlebury College

Watters, et al., 2005, Middlebury College Acknowledgments Middlebury College: Information and Library Services. Professional Development Fund provided by the Irene Heinz and John LaPorte Given Professorship in Premedical Sciences. Watters, et al., 2005, Middlebury College

The Na+/K+ Pump. I. Forwards The Movie

The Na+/K+ Pump. II. Ouabain Inhibition The Movie

The Na+/K+ Pump. III. Backwards The Movie

The Na+/K+ Pump. IV. Exchanging Na+ for Na+ The Movie

The Na+/K+ Pump. V. Exchanging K+ for K+ The Movie