Challenges and Methods in Transmembrane Protein Structure Determination Connie Jeffery University of Illinois at Chica

1. Challenges and Methods in Transmembrane Protein Structure Determination Connie JefferyUniversity of Illinois at Chicagocjeffery@uic.edu

2. Outline 1. Importance of Transmembrane Proteins 2. General Topologies 3. Methods (and challenges) for Structural Studies of TM Proteins 4. Jeffery Lab Research Interests

3. Eukaryotic cells have many membranes

4. Transmembrane Proteins • Cellular roles include: Communication between cells Communications between organelles and cytosol Ion transport, Nutrient transport Links to extracellular matrix Receptors for viruses Connections for cytoskeleton • Over 25% of proteins in complete genomes. • Key roles in diabetes, hypertension, depression, arthritis, cancer, and many other common diseases. • Targets for over 75% of pharmaceuticals.

5. Transmembrane Proteins • Cellular roles include: Communication between cells Communications between organelles and cytosol Ion transport, Nutrient transport Links to extracellular matrix Receptors for viruses Connections for cytoskeleton • Over 25% of proteins in complete genomes. • Key roles in diabetes, hypertension, depression, arthritis, cancer, and many other common diseases. • Targets for over 75% of pharmaceuticals. However, very few TM protein structures have been solved!

6. Outline 1. Importance of Transmembrane Proteins 2. General Topologies 3. Methods (and challenges) for Structural Studies of TM Proteins 4. Jeffery Lab Research Interests

7. Biological Membrane = Lipid Bilayer Approximately 30Å thick Hydrophobic core + Hydrophilic or charged headgroups Mixture of lipids that vary in type of head groups, lengths of acyl chains, number of double bonds (Some membranes also contain cholesterol)

8. Membrane Bilayer with Proteins In order to be stable in this environment, a polypeptide chain needs to (1) contain a lot of amino acids with hydrophobic sidechains, and (2) fold up to satisfy backbone H-bond propensity - How?

9. Structure Solution #1: Hydrophobic alpha-helix • Satisfies polypeptide backbone hydrogen bonding • Hydrophobic sidechains face outward into lipids

10. Examples of Helix Bundle TM Proteins PDB = 1QHJ PDB = 1RRC Single helix or helical bundles (> 90% of TM proteins) Examples: Human growth hormone receptor, Insulin receptor ATP binding cassette family - CFTR Multidrug resistance proteins 7TM receptors - G protein-linked receptors

11. Structure solution #2Beta-barrel • Beta sheet satisfies backbone hydrogen bonds between strands • Wrap sheet around into barrel shape • Sidechains on the outside of the barrel are hydrophobic

12. Examples of Beta Barrel TM Proteins PDB = 1EK9 PDB = 2POR Beta barrels - in outer membrane of gram negative bacteria, and some nonconstitutive membrane acting toxins Examples: Porins

13. General Topologies of TM Proteins Single helix or helical bundles and Beta barrels Both topologies result in hydrophobic surfaces facing acyl chains of lipids Part protruding from membrane can be a very short sequence (a few amino acids), a loop, or large, independently folding domains

14. Presence of Hydrophobic TM Domain can result in: Low levels of expression Difficulties in solubilization Difficulties in crystallization Attempting crystallization and structure solution of transmembrane proteins is considered difficult and risky.

15. Difficult and risky, but still possible:TM Proteins of Known Structure Bacteriorhodopsin, Rhodopsin Photosynthetic reaction centers Porins Light harvesting complexes Potassium channels Chloride channels Aquaporin Transporters Etc. **Although few in number, each of these structures have been important for addressing key functions.*** Great summary and resource: http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html

16. Steps in X-ray Crystallography

17. Outline 1. Importance of Transmembrane Proteins 2. General Topologies 3. Methods and Challenges a. Overexpression b. Purification c. Crystallization 4. Jeffery Lab Research Interests

18. Expression of TM Proteins Problems: Low natural expression levels Don’t always overexpress in recombinant systems Formation of Inclusion bodies

19. Expression of TM Proteins • Potential Solutions (also can help in studies of soluble proteins): • Find cell type that naturally expresses a great deal of the protein • Scale up culture sizes • Change growth conditions - temperature - 15°C, 30°C, 37°C, etc. media inducing time amount of inducing agent • Change expression vectors • Change strain or even species of expression host • Try many members of a protein family - related proteins and/or proteins from different species:

20. Methods for Solubilization and Purification of TM Proteins Problem: Hydrophobic domains tend to aggregate when taken out of the lipid bilayer - result in sticky precipitant of unfolded proteins Solution: Include mild detergent(s) in purification steps - will mask the hydrophobic regions and help solubilize the protein

21. Methods for Solubilization and Purification of TM Proteins Note: Trial and error needed to find good detergent that keeps protein folded and active Might try many detergents with different head groups And acyl chain lengths. Beta-octylglucoside = example of a common mild detergent used with studies of membrane proteins

22. Alternative Reagents for Solubilization of TM Proteins Design, synthesis, and use of: • More kinds of detergents • Detergents with novel structures (example from Prot. Science 2000, 9:2518-2527)

23. Alternative Reagent for Solubilization of TM Proteins:Lipopeptides Lipopeptides = Novel detergent/peptide hybrids (see McGregor et al., Nature Biotechnology 2003, 21:171-176) (Figures from McGregor et al., Nature Biotechnology 2003, 21:171-176)

24. Alternative Reagent for Solubilization of TM Proteins: Nanodiscs • From Steven Sligar lab at UIUC. • Goal is to put individual TM protein in environment that mimics lipid bilayer better than a micelle • Nanodiscs contain small phospholipid bilayer wrapped by membrane scaffold protein Figure from pamphlet from office of technology management, UIUC

25. Crystallization of TM Proteins Problem: Hydrophobic domains tend to aggregate when taken out of the lipid bilayer - result in sticky precipitant of unfolded proteins Solution: Include mild detergent(s) in crystallization steps - will mask the hydrophobic regions and help solubilize the protein, special screens developed for TM proteins Note: Probably need to modify lipids and/or detergents plus modifying other components of crystallization solution

26. Crystallizing Proteins

27. Additional Method for Crystallization of TM Proteins: Co-crystallization with Antibodies • Increase hydrophilic surface area • Need monoclonal Abs, and usually use fragment • Crystal contacts often between Abs Figure modified from Hunte and Michel, Current Opinion Structural Biology, 2002, 12:503-508.

28. Additional Method for Crystallization of TM Proteins: Cubic lipid phases Landau & Rosenbusch, PNAS 93:14532-14535 Nollert et al., Methods Enz. 343:183-199. • 3-dimensional lipid bilayer structure that forms in mixtures of certain lipids and water (i.e. monoolein, PNAS (1996) 93, pp. 14532-14535). • TM protein is found crossing bilayer and can interact with other copies of the protein at various angles.

29. Alternative solution for Crystallization of TM Proteins: Extramembranous Domains alone --> PDB = 2LIG • Some proteins: regions outside the bilayer are globular domains that contain the key enzymatic or binding functions. • Study these domains separate from the membrane spanning domain (using recombinant DNA techniques) • The isolated domain can often be treated like a soluble protein. • Examples - aspartate receptor, human growth hormone receptor

30. Steps in X-ray Crystallography

31. Outline 1. Importance of Transmembrane Proteins 2. General Topologies 3. Methods (and challenges) for Structural Studies of TM Proteins 4. Jeffery Lab Research Interests

32. Jeffery Lab Research Interests • Proteomics-style systemmatic study of TM protein expression • Structure and Function of Multidrug Transporters • Folding of TM proteins (Determinants of Helical Packing)

33. A proteomics level approach to TM protein studies Selection of proteins with a variety of physical characteristics and functions - Begin with study of expression and solubilization methods.

34. Cystic Fibrosis • Lethal genetic disease • 1 in 20 caucasions is a carrier • 1 in 2000 live births • Affects lungs, pancreas, sweat ducts, reproductive organs • Thick mucus secretions • Caused by mutations in the CFTR protein • Low life expectancy due in part to recurrent serious lung infections with P. aeruginosa, a multidrug resistance opportunistic bacterium.

35. A proteomics level approach to TM protein studies Clone >100 target TM proteins into similar vectors. Use constructs to test methods of expression, solubilization , purification, and crystallization. Figure modified from Gateway cloning system information from Invitrogen.

36. To be evaluated: • Do expression and membrane localization correlate with Physical features or function of the protein? Expression conditions? (including temperature, tags, vectors, strains, etc.)

37. Jeffery Lab Research Interests • Proteomics-style systemmatic study of TM protein expression • Structure and Function of Multidrug Transporters • Folding of TM proteins (Determinants of Helical Packing)

38. Multidrug Resistance • Increasing problem in medicine: bacteria becoming resistant to wide range of antibiotics • Caused by 5 major familes of transmembrane transporters (RND, ABC, MATE, SMR, MFS) • Pump many kinds of antibiotics out of cell • Info about mechanisms of functions would be useful for • finding inhibitors • finding novel antibiotics that aren’t pumped

39. MDRs of RND Protein Family Three components:Outer membrane channel + Periplasmic protein + Inner Membrane transporter Somehow the proteins work together to form a complex that crosses both membranes. The drug is accepted from the periplasm or inner membrane and transported through the outer membrane. We are working on individual proteins and complexes from Pseudomonas aeruginosa.

40. RND Protein Family Some structural information is available for individual components Three components:Outer membrane channel + Periplasmic protein + Inner Membrane transporter Reference for figure:

41. RND MDR Family • Additional structures and biochemical/biophysical characterization would help with: • How do the 3 protein components fit together? • How is proton motive force used to pump drugs? • What is path of drugs through protein? • How do inhibitors inhibit the pumps? • How do the different RND transporters select different subsets of drugs? • What compounds (novel antibiotics) would escape pumps?

42. Jeffery Lab Research Interests • Proteomics-style systemmatic study of TM protein expression • Structure and Function of Multidrug Transporters • Folding of TM proteins (Determinants of Helical Packing)

43. Protein Folding Problem How does a one-dimensional amino acid sequence determine a specific three-dimensional structure? Or How can we read the sequence and predict that structure?

44. General Idea We know what an alpha-helix or a beta strand looks like, so (1) figure out which parts of the sequence are helices and which parts are strands (2) figure out how they pack together For soluble proteins, neither is well predicted. But for transmembrane proteins ...

45. TM Protein Structure Prediction, Step #1 For alpha-helical transmembrane proteins, hydropathy plot analysis provides a fairly accurate method to predict which amino acids form membrane-spanning helices We can model the structure of an individual alpha helix fairly accurately.

46. TM Protein Structure Prediction, Step #2 • How do the helices pack in the membrane? There are several labs studying known protein structures to identify factors involved in determining how transmembrane helices pack together (specificity of interaction and packing motifs) Hydrogen bonds Hydrophobiciity Amino acids known to face the lumen of a channel Multiple sequence alignments Helix packing sequence motifs, etc. These kinds of information are then combined with protein docking and energy minimization programs to predict how the helices pack together. • It is quite possible that studies of helical transmembrane proteins could lead to key information about the protein folding problem - how to predict protein structure from amino acid sequence

47. Summary • Transmembrane Proteins play many important processes in cellular processes in both health and disease • Two general type of tertiary structure are found to cross the membranes: beta-barrels and alpha-helices • Structural Studies of TM Proteins are impeded by difficulties in overexpression, purification and crystallization • However, the few dozen structures that have been determined have provided key information about channels (gating, selectivity, etc.), energetics, transport, and other transmembrane processes • Analysis of helical transmembrane protein structures may lead to accurate predictions of protein structure from amino acid sequence for this type of protein

48. University of Illinois at Chicago Graduate Studies in Biology The Department of Biological Sciences at UIC provides training leading to the Ph.D. degree in Molecular, Developmental and Cellular Biology. Full tuition waiver & competitive stipend available for qualified candidates. For more information visit http://www.uic.edu/depts/bios.

49. UIC Dr. Joseph Orgel Diana Arsenieva Ji Hyun Lee Forum Bhatt Kathy Chang Vishal Patel Bong Bae Vidya Madhavan Ryo Kawamura Financial Support UIC Campus Research Board UIC Cancer Center/American Cancer Society Cystic Fibrosis Foundation American Heart Association American Cancer Society Acknowledgements