Studying dynamic protein:protein interactions in vesicle trafficking. David Owen CIMR University of Cambridge

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1 Studying dynamic protein:protein interactions in vesicle trafficking David Owen CIMR University of Cambridge

2 The importance of vesicle trafficking in mammalian cells Transducing signals small molecules/nutrients Cell/substratum and Cell/cell conatcts Enzymatic activity Pathogens

3 Life of transport vesicle Resident proteins Disassembled coat Vesicle coat Cargo and SNAREs Motor Tether Cytoskeleton 1 Coat assembly 5 Transport/targeting 6 Coat disassembly 2 Cargo and SNARE selection to acceptor membrane 7 Proofreading by rabs 3 Mechanical deformation 8 SNARE mediated 4 Scission of vesicle. fusion with target Clathrin-coated vesicles are the most common post-golgi transport vesicles and include those involved in clathrin-mediated endocytosis from the cell surface

4 Endocytic clathrin-coated vesicles forming in vivo GFP clathrin Using TIRF x36

5 Anatomy of a clathrin-coated transport vesicle Vesicle coats often contain 20+ different soluble proteins that are recruited from cytosol to the membrane in a spatially and temporally regulated fashion Coat formation involves many protein:protein, protein:phospholipid interactions all of which are dynamic and readily reversible with um K D s so ideal for ITC but we also use SPR!!!!! Clathrin-coated transport vesicle Clathrin scaffold linked to cargo and the membrane by clathrin adaptors

6 Studying the recruitment of coat components to liposomes using SPR Collaboration with Stefan Hoening (Cologne) Mimic protein recruitment to multi-component membranes Liposomes of defined lipid composition containing peptides corresponding to cytosolic portions of cargo motifs attached to lipidic tails via maleamide/cys coupling with folded docking proteins attached ot the surface Fluorimetry and ITC are problematic due to size of AP2 ( kDa) and effects caused by the presence of liposomes and weak K D s<200um We use SPR with liposomes immobilised on sensor chips via dextran-linked hydrophobic acyl chains sample 100 nm dextran layer 50 nm Gold layer

7 Structural characterisation of protein:protein interactions Identify candidate protein:protein interaction from Y2H screening, immunoprecipitations and/or tagged pull downs followed by mass spec. Make whole or relevant domains recombinantly and us these to confirm existence of and to further map the interaction Use ITC to characterise the strength of interaction most K D um range and chose appropriate strategy: If K D 100nM to 1-5uM then mix and copurify on gel filtration for xtalisation If K D 5-100uM and peptide mediated use large excess of synthetic peptide in xtalisation trails If K D 5-100uM and uses folded epitopes link together at DNA level to artificially increase concentration (often get intermolecular interactions) If K D >200uM NMR shift mapping is likely your only hope

8 Vesicle coat protein : SNARE interactions

9 Interactions in vesicle transport Resident proteins Vesicle coat Components Disassembled coat SNARE mediated fusion with target Cargo and SNAREs Motor Tether Cytoskeleton If SNAREs are to be incorporated into vesicles (both to allow their fusion with target membranes to occur and to allow the return other SNARE to their steady-state cellular localisations), then there must be active recognition of the cytosolic portion of SNAREs by various vesicle coat proteins

10 SNAREs Membrane anchored : type II integral membrane proteins or lipiated Involved in membrane fusion events in vesicle trafficking and organelle biogenesis 38 SNAREs in the human genome (~25 expressed in each cell) with specific subcellular distribution in different organelles and carriers. N-terminal 16 turn helical Domain SNARE motif TM lumenal or lipid modification SNARE complex formation Contain at least one amphipathic 16 turn amphipathic helical SNARE motif, four of which come together to form coiled/coil SNARE complexes N-terminal domain, which varies in size ( residues) and structure

11 SNARE hypothesis of membrane fusion. SNAREs provide energy and specificity to membrane fusion events Energy derives from tight SNARE complex formation pulling the membranes close enough together to facilitate merging of membrane bilayers : Requires spatial and temporal regulation of SNARE fusogenic activity Trans-SNARE complex Membrane(s) Post-fusion Cis-SNARE complex Post fusion cis-snare complex Brunger 1998 Specificity derives from several sources and is more complicated Limited combinations of 3Q- and 1R- can form productive SNARE complexes. SNAREs subcellular distribution patterns imply active/selective SNARE transport Tethers and SNARE regulators binding small G-proteins, PIPs also contribute

12 Some examples of SNARE:coat recognition VAMP7:VARP Ingmar Schaeffer and Lauren Jackson VAMP8:CALM Sharon Miller

13 Trafficking of the main post Golgi R-SNAREs Lysosome and LROs Early endosome Late endosome recycling endosome VAMP7 functions on late endocytic compartments. Needs to be endocytosed and trafficked through the early endocytic pathway in an inactive form VAMP3/8 are needed in a potentially active form i.e. uncomplexed so that they can function early on the endocytic pathway Longin domain VAMP7 VAMP3 Late endosomes with lysosomes LROs and the plasma membrane plasma.membrane <-> endosomes VAMP8 endosome <-> endosome and p.membrane <-> endosome

14 VAMP7 is a late(ish) endosomal SNARE Largest (120 residue folded longin domain) and the ancestor endocytic R-SNARE SNARE motif Steady state localisation late endosomes, endolysosomes and lysosomes : Colocalising with late endocytic markers lpg120/lamp1, Rab7 in HeLa and NRK N TM helix C Involved in fusion of late(ish) endosomes and carriers derived from them with endolysosomes, lysosomes, cell surface and autophagosomes To identify potential binding partners, full cytoplasmic domain and N-terminal longin domain were used in Y2H library screens

15 VAMP7 binds to VARP VARP only identified in a Y2H screen using the full cytosolic portion of VAMP7 and not in longin domain screen confirmed by GST pulldowns VARP is a 1050 residue multidomain Rab21/Rab5 GEF and Rab32/Rab38 effector VARP is ubiquitously expressed and highly enriched in melanocytes and ha been proposed as being is involved in dendrite and neurite outgrowth In HeLa and NRK cells VARP localises to late endocytic compartments and also binds to the vesicle coat retromer, which is involved in transport from endosomes 1 VPS9 GEF domain Rab21 Rab5 Zn Retromer ANKRD1 Rab32 Rab38 Zn VAMP7 ANKRD VAMP7 binding mapped to N-terminal of the second set of ankyrin repeats K D 3uM

16 Measuring K D for VARP ANKRD2 binding VAMP7 Binding tight enough to try making complex in solution and purifying by gel filtratiion Complex co-eluted from gel filtration and set up into crystallisation trials

17 Structure of VAMP7 bound to ANKRD2 domain of VARP GEF domain ANKRD1 ANKRD2 Zn Zn 1 Unpredicted ankyrin repeat Rab21 Rab32 VAMP7 ANKRD VAMP7 SNARE motif VAMP7 longin domain Interface composed of parts of both longin domain and SNARE motif

18 Analysing mutations in VARP:VAMP7 interface by ITC A mutant VARP M684D,Y687S (MY) was designed&created that cannot bind VAMP7 A mutant VAMP7 (DES) was designed&created that cannot bind VARP Both mutants were used in subsequent in vitro and in vivo functional studies

19 VARP inhibits VAMP7 based SNARE complex formation SNARE motif trigger Trapped by VARP ANKRD2 SNARE complex formation initiated at the N-terminus of the SNARE motif trigger Trigger is bound by VARP : WT VARP can inhibit VAMP7 SNARE complex formation MY mutant VARP cannot bind VAMP7 so doesn t inhibit SNARE complex formation

20 Trafficking of the main post Golgi R-SNAREs Lysosome and LROs Early endosome Late endosome recycling endosome VAMP7 functions on late endocytic compartments. Needs to be endocytosed and trafficked through the early endocytic pathway in an inactive form VAMP3/8 are needed in a potentially active form i.e. uncomplexed so that they can function early on the endocytic pathway Longin domain VAMP7 VAMP3 Late endosomes with lysosomes LROs and the plasma membrane plasma.membrane <-> endosomes VAMP8 endosome <-> endosome and p.membrane <-> endosome

21 CALM is involved in R-SNARE endocytosis Genetic interactions in several organisms implicated homologues of the 60kDa+ clathrin adaptor CALM (Clathrin Assembly Lymphoid Myeloid-leukaemia protein) in the endocytosis of small endocytic R-SNAREs. CALM has a 30kDa, N-terminal ANTH domain, which binds PtdIn4,5P 2 At the plasma membrane and a clathrin-binding C-terminal tail. Clathrin binding Mutations in CALM cause growth retardation, anaemia and shortened life span in mice. CALM has been implicated in causing cognitive defects with ageing and as a risk factor for developing Alzheimer s in humans. ITC and GST-pull downs show that mammalian CALM ANTH domain binds the SNAREs VAMP3 and VAMP8 with K D s of ~20µM ANTH PtdIn4,5P 2 Co-recognition of VAMP with PtdIns4,5P 2 (K D also ~20µM ITC) results in an apparent K D for the interaction with a membrane containing both VAMP8 And PtdIn4,5P 2 to ~0.2µM (Liposome-based SPR)

22 Binding of CALM to small endocytic R-SNAREs ITC and GST pull downs narrowed binding site down to residues of VAMP8 i.e. the first half of SNARE motif that forms an amphipathic helix

23 Surface of CALM ANTH has a large hydrophobic trough Attempts to crystallise a complex of CALM ANTH and VAMP8 yielded only numerous crystal forms of unliganded ANTH domain (residues 1-289) 264 Resolution 1.8Å R/R free 18.2/ Increasing Hydrophobicity Residues often a poorly ordered helix that sits in a hydrophobic trough Since SNAREs are hydrophobic and helical, could this be the VAMP binding site? In an attempt to increase affinity for VAMPs, the potentially autoinhibitory part (residues ) were removed...

24 Truncated CALM is a dimer and doesn t bind VAMP8 Resolution 1.7Å R/R free 17.0/19.1 MALS indicated CALM residues formed an obligate dimer Structure of the dimer showed the dimer interface was the proposed VAMP binding site VAMP8 SNARE motif was coupled to the C-terminus of CALM ANTH in place of residues

25 Structure of CALM ANTH :VAMP8 Complex CALM ANTH - helix 11 linker VAMP Resolution 2.0Å R/R free 18.2/19.9 In order to bind the SNARE helix displaces the final helix of the ANTH domain

26 Details of the CALM ANTH :VAMP8 complex interface CALM ANTH - helix 11 linker VAMP810-41

27 Mutating residues in the interface abolishes the CALM ANTH :VAMP8 interaction

28 The K24AM27A mutant VAMP8 is not endocytosed Anti HA Uptake Assay Wt VAMP8HA can bind CALM and is largely endosomal K24A,M27A VAMP8HA can t bind CALM and is largely on the cell surface

29 Mutant versions of CALM which cannot bind VAMP8 do not support VAMP8 endocytosis Wt VAMP8HA uptake VAMP8-HA cell line transfected with RNAi resistant CALM wt, L219S or M244K Cells mixed with untransfected control cells Endogenous CALM depleted by sirna Wt CALM L219S CALM M244K CALM In RNAi Resistant CALM wt transfected cells VAMP8HA is endocytosed In RNAi Resistant CALM L219S and M244K transfected cells VAMP8HA is not endocytosed

30 In conclusion ITC, backed up by GST pull downs is invaluable in proving the existence of protein:protein and protein:phospholipid headgroup interactions. Characterisation of strength of interaction by ITC allows appropriate strategies for crystallisation to be designed ITC, again backed up by GST-pull downs using mutant versions of proteins allows sites and mode of interactions to be confirmed In conjunction with liposome-based SPR, ITC allows the more complex interactions in multicomponent membrane-based systems to be analysed. Together these data can be used to probe the role of interactions in complex in vivo systems.

31 Acknowledgements VAMP7:VARP Collaboration Ingmar Schaeffer (MRC-LMB now Max Planck Martinsried) Lauren Jackson (CIMR now Vanderbilt, USA) Paul Luzio (CIMR) Geoff Hesketh (CIMR now University of Toronto) VAMP8:CALM Collaboration Sharon Miller (CIMR) Stephen Graham (CIMR now University of Cambridge) Andrew Peden (CIMR now University of Sheffield)

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