Quaternary structure / Protein interfaces JBB2026H. Lecture 2: Protein Structure II. Most proteins are oligomers

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1 G. Privé Sept 2014 JBB2026H Lecture 2: Protein Structure II 1 Quaternary structure / Protein interfaces Most proteins are oligomers Common 3 structure does not imply common 4 structure Most oligomeric proteins are not stable as monomers - think about the free energy of the monomer - can we describe the single chain folding of a protein with 4 structure purely at the 3 level? Subunit interfaces are often used as functional hotspots protein-protein interactions are a type of quaternary structure. 2

2 Quaternary structure: More than 80% of proteins are oligomeric heterooligomers homooligomers <12 oligomeric state Goodsell and Olson Ann. Rev. Biomol. Struc.29, 105 (2000) 3 The glycolytic enzymes PDB molecule of the month Feb

3 Diversity of BTB domain self-association Skp1 BCL6 Schulman, B.A., et al. (2000) Nature 408: 381 Ahmad, K.F., et al. (2003) Mol. Cell 12: 1551 Kv4.2 GAGA factor Zhou, W., et al. (2003) PNAS 100: 8670 Ghetu, A.F., et al. To be Published 5 Interfaces in oligomeric proteins geometric and electrostatic complementarity Generally multiple, weak contacts (large surface area). Shape complementarity (VdW) Additional stabilization: disulfides, metals, cofactors,... Huge variety - cannot make many generalizations Range of affinities/exchange rates: stable (low Kd, generally has a hydrophobic character) vs. transient or exchangeable (higher Kd, usually more hydrophilic intermolecular surface). Individual exchange rates (kon, koff) do not necessarily correlate with affinity (Kd) (but Kd is given by their ratio koff/kon) Stable: usually shielding many hydrophobic groups - Net result is a very strong association. (i.e. these proteins typically exists exclusively in the oligomeric form) These interfaces are often indistinguishable from the interior of proteins Transient or exchangeable: usually more polar, often contain bridging waters buried surface typically : Å**2, polar, 0-10 H-bonds, bridging waters 6

4 2:2 complex of the BCL6 BTB domain with the SMRT BBD peptide Ahmad et al. Mol Cell (2003). 7 2:2 complex of the BCL6 BTB domain with the SMRT BBD peptide 8

5 9 Advantages of oligomeric proteins Evolutionary advantage - Symmetric oligomers are more fit Large size Oligomeric Symmetrical Functional Genetic Physicochemical Larger proteins more stable against denaturation (smaller surf/vol) More efficient use of intracellular water Bigger is better - but limitations of protein synthesis Error control Coding efficiency Stability of association Multivalent binding / allostery Self assembly Goodsell and Olson Ann. Rev. Biomol. Struc.29, 105 (2000) 10

6 Examples of Point Group symmetries Chiral point groups in 3D - the operation does not change the object - rotations only (no reflections or inversions) - rotations axes intersect at a point - results in repeated, equivalent surfaces between chains - generates closed structures Cyclic (Cn) n=2,3,4,... rotation about a single axis Dihedral (Dn) rotational symmetry about one axis, and C2 rotations perpendicular to the n-fold axis (results in n Cn axes) Cubic T: tetrahedral (C2 and C3 axes) O: Octahedral (C4, C3 and C2 axes) I: Icosahedral (C5, C3 and C2 axes) 11 Line Group symmetry (helical symmetry) Combination of rotational and translational (1D) symmetries Leads to fibrous structures. Combine rotation with translation along axis (screw rotation) Produces supermolecular helices e.g. Actin, microtubules, flagella, Often used in structural/architectural roles open structures - how to terminate the chain? need to cap the ends Keratin rare (biologically): plane group (2 translations), space group (3 translations) 12

7 Generation of Oligomers by 3D Domain Swapping mechanism for the formation of new interfaces results in higher order quaternary structures requires only small changes in a hinge loop Usually involves a C or N terminus 13 Domain swapping Liu and Eisenberg, Protein Science 11, 1285 (2002). 14

8 Membrane Proteins Some general rules for understanding membrane protein structure: - Satisfy the main-chain hydrogen bonds - transmembrane alpha-helices - beta barrels - No unsatisfied H bonding groups exposed to lipid - Match the hydrophobic properties of the side chains and lipids -! align protein surfaces with the hydrocarbon chains Exceptions occur! 15 Most membrane proteins can be classified into one of two structural classes Very few exceptions known so far, but there are many variations to the theme. Proteins are complicated! Alpha-helical bundles Single or Multiple passes through the membrane Bundles with ~ aligned helices Commonly find cofactors Beta-barrels antiparallel beta-sheet the last strand is H-bonded to the first strand to close the barrel. This solves the edge problem and satisfies all main chain H bonding. 16

9 Membrane proteins Low dielectric constant within the bilayer No water in the middle of the bilayer Main chain fully H-bonded (2 structure) - α-helices: local (i, i+4) H-bonding - β-barrels: H-bonding between widely separated parts of the chain TM region: Simpler topologies (3 structure) than soluble proteins α-helix bundles ; β-barrels Side chains point outward from helices or barrels energetics and folding pathways are very different than for soluble proteins Many MPs have both a membrane domain and a soluble domain 17 The membrane presents a very complex environment - and there are many types of membranes! Bulk solvent 15 Å Headgroup region 30 Å Hydrocarbon region 15 Å Headgroup region Bulk solvent 18

10 Contributions to the free energy of folding for membrane proteins are different (and often more complex) than for soluble proteins The cost of unformed H-bond in a membrane is very high No competing water in the bilayer MPs largely limited to TM helices and beta-barrels. Simpler topologies The dielectric constant changes from 2-4 within the bilayer to ~80 on the outside surface. charged and H-bonding groups are driven away from the lipid phase. There is essentially no water in the middle of the bilayer - no hydrophobic force to drive 3 structure! - Helices pack in a hydrophobic environment What holds them together? MP structure is not driven by the effects of water - van der Waals (packing) term is important 19 Composition of TM helices Rich in hydrophobic amino acids (Leu, Val,...). Trp and Phe often observed at the interfacial region. Glycine, proline and cysteine are fairly common. But no disulfide bonds Sometimes find polar residues (H-bonding between TM helices) Salt bridges can occur. Channels, pores, transporters, etc. often have polar interiors. Polar amino acids in a TM helix often point in. Functionally important. 20

11 Residue distributions in alpha helices 24% charged 6% charged 33% phobic Y 3 M 2 V 6 I 6 C 2 P 5 L 9 F 4 W 1 T 6 S 7 D 5 A 7 E 6 Q 4 K 6 N 5 H 2 R 5 G 7 41% polar 54% phobic L 16 I 10 Y 3 F 8 W 3 M 4 V 8 DE K H C 2 R 1 P 4 G 8 T 7 N 3 Q 2 S 7 A 9 40% polar soluble proteins Transmembrane (TM) domains in membrane proteins F,L,I make up over 1/3 of the residues in TM helices 21 Example of an α-helical membrane protein: ABC transporter α-helical membrane proteins occur in: - eukaryotes: plasma membrane, most organelle membranes, inner membrane of the mitochondria - bacteria: cell membrane (Gram positive); inner membrane (Gram negative) 22

12 Rules are sometimes broken! The pink helix goes only halfway through the TM region! GlpF - Glycerol facilitator allows diffusion of glycerol KcsA - potassium channel - reentrant loop forms the selectivity filter 23 some beta-barrel structures: n=3x4=12 n=8 n=22 n=12 n=16 Present in: - outer membrane of Gram -ve bacteria - outer membrane of mitochondria 24

13 Saposin proteins Family of four ~80 amino acid α-helical, cysteine rich lysosomal proteins SapA, SapB, SapC, SapD Non-enzymatically active but found to be essential in the hydrolysis of specific glycosphingolipids Also essential in CD1 lipid-antigen presentation in the immune system Belong to a family of saposin-like proteins (SAPLIPs) Exist in both soluble and membrane-bound states Saposin functions are based on reversible lipid interactions 25 Saposin activator proteins: Role in the catabolism of galactosylceramide galactose Krabbe Disease is caused by a deficiency in the ability to catabolize galactosylceramide 26

14 Two models for saposin activation of the hydrolysis reaction Saposin proteins function via reversible lipid interactions saposin hydrolase solubilization (detergent-like activity) liftase apo state lipid complex state Target lipid bilayer membranebound state 27 Structures of saposins A, B, C and D (apo state) Saposin A Saposin B Saposin C Saposin D Ahn V. E. et al. PNAS 2003, Protein Science. 2006; Popovic and Privé Acta D

15 SapB dimer apo lipid-occupied 29 30

16 Buried surface at the SapB dimer interface apo lipid-occupied protein-protein contacts in the dimer 31 Saposin A forms monodisperse 3.2 nm protein-lipid complexes 2 protein chains and 8-12 lipids +PC +lipid mix apo Size exclusion chromatography Dynamic light scattering Popovic et al., PNAS (2012) 32

17 1.9 Å structure of Saposin A co-crystallized with LDAO SapA adopts an open form N N C C N C inner hydrophobic surface outer hydrophilic surface Popovic et al., PNAS (2008) 33 Hinge opening in Saposin A 34

18 Hinge opening in Saposin A 35 SapA homodimer with 40 LDAO molecules 36

19 Top Bottom SapA homodimer with 40 LDAO molecules (~30 kda complex) 37 Similarities with discoidal HDL particles and recombinant HDL particles (nanodiscs) capped bilayer structures can be used to provide a natural bilayer environment to a solubilized membrane protein 5.5 nm 4 nm 10 nm 160 phospholipids 2x apolipoprotein AI Shih et al., Journal of Structural Biology (2007) 5 nm 8-12 phospholipids 2x SapA Popovic et al., PNAS (2012) 38

20 Acyl chain packing in the saposin A core Lateral chain packing in lipids and membranes Donald M. Small, Journal of Lipid Research (1984). hexagonal saturated hydrocarbon chain packing 21 Å2 / acyl chain 39 Coarse-grained molecular dynamics of SapA / DOPC supports the crystal model John Holyoake, Régis Pomès 40

21 Solubilization model for the saposin A activation reaction - detergent-like activity Hydrolase enzyme Saposin A lipid complex state apo state membranebound state Target lipid 41 SapA Monomer in absence of lipids, dimer as a lipid complex Forms small, stable 3.2 nm complexes with a wide variety of lipids Crystal structure with LDAO reveals a discoidal assembly with 40 internalized detergents surrounded by two SapA molecules Inside-out structure relative to integral membrane proteins No direct protein-protein contacts in the complex - the assembly is driven by the sequestration of the lipid/detergent acyl chains No dyad in the plane of the disc, resulting in an asymmetric bilayer Supports a solubilizer model for the Galactosylceramidase activation reaction 42

22 SapB Stable homodimer - formed by stable Sap-Sap contacts flexibility within the dimer (limited) lipid cavity for internalization of 2-3 lipids solubilizer model SapC Stably bound to membranes liposome aggregation brings enzyme (GalCer-ase) to remodelled regions Liftase model 43 44