Protein Structure Realities: Insights into Function, Classification, and Folding

1. Protein Structure Realities Andy HowardBiology 5553 October 2016; revised 11 Oct 2018

2. Protein structure matters! • We care about protein structure because it tells us about function, and because it enables us to classify proteins and identify features and functions • This discussion is independent of the tools used to determine the structure.

3. Agenda • Helices • Sheets • Turns • Structural classes • Intrinsically disordered proteins • Protein folding: statics • Protein folding: dynamics • Structure-function relationships • Enzymes • Immunoglobulins • Receptors

4. Alpha helices • H-bonds: NH .. O=C of residue 4 farther on • 1.5Å rise /residue * 3.6 residues/turn =5.4Å per turn = pitch • Diameter ~ 6Å not counting side-chains • φ -80º to -50º • ψ -70º to -30º Cartoon courtesy knowledgeserver.wordpress

5. Properties of the alpha helix • Main-chain H-bonds are parallel and face the same direction • So there’s a net dipole moment associated with any one helix along the axis • Side chains emerge almost perpendicular to the helix axis, minimizing steric hindrance • First few & last few residues in a helix miss some H-bonds

6. Ramachandran plot: myoglobin • Non-helical residues are at the turns.

7. Other helices • 310 helix • 3 residues (10 atoms)/turn • H bond is 3 residues away, not 4 • Sometimes found at one end of an otherwise alpha helix • π helix • 4.4 residues (16 atoms) /turn • H bond is 5 residues away • Pretty rare

8. β sheets • Main-chain H-bonds still-NH … O=C (why?) • No particular distance along the chain between H-bonded atoms • Extended chain: almost, but not fully, flat • Side-chains alternate up, down sandwalk.blogspot.com

9. Relationships between strands • If two adjacent strands run the same direction (e.g., left-to-right is N to C) then they’re parallel • If they run in opposite directions (left-to-right is N to C in one and C to N in the other) then they’re antiparallel • Both are common and have a few different properties

10. Antiparallel sheets • 2-20 strands arranged in anti-parallel fashion • Hydrogen bonds almost perfectly straight • Pleating of sheet is modest • Note alternating 2/4 atom patterns • Inter-strand distance 0.347 nm • Often amphiphilic – alternating polar, non cryst.bbk.ac.uk

11. Parallel sheet • 2-20 strands arranged parallel (usually > 4) • Hydrogen bonds significantly bent • Pleating more pronounced (straightens the H-bonds somewhat) • Strands 0.325 nm apart • Typically have hydrophobics on both sides

12. Ramachandran angles for strands • Typically -160 < φ < -70, 95 < ψ < 160 • Antiparallel sheets are more flexible—wider range of plausible values • Parallel concentrated near φ= -120, ψ=100

13. Ramachandran plot: TIM • More complex than I expected!

14. Beta turns • Transitions from 1 antiparallel beta strand to next • Carbonyl oxygen forms H-bond with main-chain H-N of residue 3 positions down • This stabilizes the turn and almost maintains the beta hydrogen bonding pattern • Prolines tend to force beta turns; glycines allow them Loren Williams, Georgia Tech

15. Structural classes • Most globular proteins are in one of these four classes: • Antiparallel α-helix proteins • Parallel or mixed β-sheet proteins • Antiparallel β-sheet proteins • Metal- and disulfide-rich proteins • Combinations sometimes appear, e.g. one type in each domain

16. Antiparallel α-helix proteins • Simple way to pack helices: antiparallel • Usually:15º left-handed twist of the bundle • Sometimes one helix tilts away from bundle • Globins: 2 perpendicular layers, chain moving back and forth between layers

17. Parallel or mixed β-sheet • Typically found in the core of a protein with little solvent access: so hydrophobic • A common instance: an 8-stranded parallel β-barrel inside a set of connecting helices: the TIM barrel(10% of enzymes!) C&E News

18. Parallel-sheet proteins, continued • Another type: doubly wound parallel β-sheet: imagine strands wound from the middle in both directions, protected by helices or other substructures Flavodoxin - Wikimedia

19. Antiparallel β-sheet proteins • Simplest is two layers (sandwich): hydrophobics inward, hydrophilics outward • Often wrapped into a cylinder or barrel Soybean trypsin inhibitor – intechopen.com

20. Special case: Greek Key • Interlocking topology like patterns found on ancient Greek vases • Many examples – often with an extra motif

21. Metal & disulfide-rich proteins • Typically: small proteins, with metals and disulfides providing much of the enthalpic drive toward folding • Often look like small, distorted versions of the 3 previous classes (but not insulin or crambin) Phospholipase A2 crambin

22. Intrinsically disordered proteins • Some proteins don’t adopt a defined 3-D structure under any biological conditions • More common are proteins that are disordered in the absence of ligands or protein partners • Typically they contact their partners over a wide surface of interaction • Generally hydrophilic: lots of EKRGQSP, less ILVWFYCN Dyson & Wright Nature Rev. Mol. Cell Biol. 6, 197-208 (March 2005)

23. Protein folding: statics • We’ve discussed this already: this is just a quick review • Critical experiment by Anfinsen showed that many proteins can be reversibly denatured • That means that the folded state is a thermodynamic energy minimum and there exists a kinetic pathway that leads to the folded state in a finite amount of time • Consider Levinthal’s paradox!

24. Protein Folding: Dynamics • Generally believed that full 3-D ordering is the outcome of a multi-step process with partially folded states in the middle • Isolated secondary structures probably fold up first (helices, strands, partial sheets) • Then those organize themselves into larger tertiary structures

25. Folding according to Kinemage

26. Structure-function relationships • It is a truism that structure determines function, but what does that really mean? • Briefly: for a protein whose overall function is known, the structure will tell us how it does its job • For a protein whose function is initially unknown, the structure may (usually by analogy) tell us what it does… and then how it does it. Tran EEH, Borgnia MJ, Kuybeda O, Schauder DM, et al. (2012) Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation. PLoS Pathog 8(7): e1002797. doi:10.1371/journal.ppat.1002797 http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002797

27. Pharmaceutical applications • If you know the structure of a protein that is associated with a disease state, you can use that structure to help you design inhibitors of that protein and thereby influence the disease state • This can be iterative: • Use the apo-structure to design the first inhibitors • Use the protein-inhibitor complex to tell you how to modify those inhibitors to get better binding or (maybe) better pharmacokinetics

28. Generalizations about structure • We want to go a bit beyond hand-waving about what kinds of structures are associated with particular functions • Recognize that some motifs are closely associated with very specific functions (e.g. NAD binding) whereas others are more versatile (TIM barrels)

29. Enzymes • Generally the active site is internal • Even if the substrate is polar, the process of immobilizing it and activiting it typically occurs in a mostly nonpolar environment • Therefore enzymes tend to be approximately spherical with a channel, perhaps dynamically switched, leading to the internal active site

30. Immunoglobulins • Also roughly spherical, but the similarities end there • The entire job of an immunoglobulin is to recognize and bind to an extended surface of a protein or ligand • Therefore its functional domain (the CDRs) is spread out and can be fairly planar • The important stuff is on the outside, not the inside! CD3εγ and OKT3 Fab

31. How to characterize the antigen-binding interaction • Typically the energetic driver for binding is the complementation or covering of exposed hydrophobic surfaces. • That is, the amount of hydrophobic surface area on the antibody that is buried by the binding event will determine the binding constant • Very small Kd values don’t necessarily correlate with the utility of a particular antibody; sometimes important antibodies have multi-micromolar Kd’s.

32. How does this work structurally? • Interactions occur in the variable light (VL) and variable heavy chain (VH) regions • Both can contribute to the antigen-antibody interaction, although the VH interactions are typically more critical. • Typically there are three complementarity determining regions (CDRs) for any given antigen; thus 6 sequences per antigen

33. Typical Fab for a small-molecule antigen • 4-4-20 anti-fluorescein Fab • CDRs can be identified because the structure was determined with the antigen bound • Often the antigen-bound form is considerably more stable than the apo form

34. Receptors • Most are 7-transmembrane helical proteins, and even the ones that aren’t look somewhat like the 7-THPs • Needs to have appropriately nonpolar helical regions • Extracellular and cytosolic domains are typically small and somewhat polar • There has to be a signaling capability! NTS1, a GPCR; Anthony Watts

35. GPCR specifics • Generally 7-THPs • N-terminus is extracellular,C-terminus intracellular • Ligand-binding domain is typically in the second extracellular loop, except when the ligand is big • Binding of ligand causes a twisting of the transmembrane helices relative to one another • Interaction with G protein is generally at the C-terminus

36. GPCR set in lipid raft