Biol2012 Exploring Proteins PDF

Biol2012 Exploring Proteins Jörn Werner  Today’s Lectures: Case Study 1. Introduction into HIV life cycle 2. Introduction into HIV viral assembly and maturation 1.Consider two distinct states: immature and mature 3. GAG modular protein, 4. Divide and conquer 1.X-...

Biol2012 Exploring Proteins Jörn Werner  Today’s Lectures: Case Study 1. Introduction into HIV life cycle 2. Introduction into HIV viral assembly and maturation 1.Consider two distinct states: immature and mature 3. GAG modular protein, 4. Divide and conquer 1.X-ray and NMR structure of GAG domains 2.Shape of full length GAG using scattering 5. Electron microscopy to study assembled viral particles 6. Viral assembly and structural plasticity 7. Example of interface plasticity in C-CA domain selfinteraction Live cycle of HIV 1 World wide 40 million infected individuals no cure, no vaccine, Current treatments help to control virus  Assembly & maturation of HIV-1 HIV protease cleaves assembly of unprocessed Gag into domains GAG into the immature MA,CA, NC virion causing maturation of (non-infectious) the virus particle (infectious) Gag-proteins Gag shell MA Gag clevage CA by HIV- 1 NC proteas RNA e  GAG is a modular protein MA NCA CCA NC P6 Immature Self trimer Self hexamer Self RNA TSG101 dimer mature Self trimer Self hexamer Self RNA TSG101 dimer 5 HIV1 protease cleaves Gag in 5 places 1 2 3 5 4 P6 MA SP1 SP2 CA NC  Dissect and conquer: Matrix - forms trimes - Associates with the membrane via myristol modification  Excursion: NMR and x-ray structures 1) NMR structure of Matrix (MA)N-terminus Overlay of 20 structures In the helical core all structures overlay well, but N- and C-termini show large variations C-terminus PDB: 2HMX NMR produces a family of structures that are consistent with pair wise distance constraints (derived from proton-proton interactions) No constraints means structure is not defined (see N- and C-termini above)  Excursion: NMR and x-ray structures Comparison of X-ray and NMR structures of MA Ile19 - Removed the first 7 and last 22 residues in solution structure since Gln28 no x-ray structure available Val8 - Mean difference of Ca atom positions is less then 1 Å - Two loop regions show larger differences: Ser72 - Ile19-Gln28: NMR multiple structures but different from x-ray - Leu68-Ser72; NMR well Leu68 defined structures different from x-ray PDB: 2HMX (blue) and 1HIW (red) Excursion: NMR and x-ray structures MAA Difference around Ser72 because in the Val8 crystal MA forms trimers (MAA,MAB,MAC) and Ser72A the region around Ser72C Ser72 is in the trimer interface The region between Ile19-Gln28 is surface Ser72B MAexposed (dynamic?) MAB C  Excursion: NMR and x-ray structures General feature of the differences between solution and crystal structures – Disordered regions of proteins do no diffract and hence x-ray structure not defined (e.g. here N and C-termini) – Core structures agree well between x- ray and solution state – Loop regions may be different because of packing interactions in crystal ( eg ~Ser72) because in solution these regions are flexible (Ile19-Gln28) Dissect and conquer: Capsid After cleavage of matrix domain (mature form of capsid) N-terminal loop of capsid (yellow) folds back on itself as shown here. N-CA In immature GAG (when matrix and capsid are covalently linked) the N- terminus of capsid is extended and forms a flexible linkage with the matrix domain Individual structures of N-CA and C- C-CA CA determined by x-ray and NMR N-CA and C-CA domain linked flexibly (hence no x-ray structure). inter domain flexibility determined by NMR  Dissect and conquer: MA- NCA 2 views of 20 NMR structures of MA- NCA MA MA NCA NCA Overlaid on NCA Overlaid on MA Conclusion: both domains are well defined and the inter-domain linker is flexible Nucleocapsid (NC) binds (viral) RNA NC Zn2+ knuckle fold Psi packaging region of HIV RNA interaction site for NC Interdomain linkage regions SP1 SP2 MA to NCA: flexible linker (see slide 22) NCA to CCA: flexible linker (data not shown) SP1 region: under investigation: some measurements suggest single helical structure others suggest flexible SP2 and p6: flexible regions (with recognition sites for host proteins) Shape of full length GAG using scattering RG: radius of Gyration: size of the molecule Scattering Intensity Scattering angle Shape of full length GAG: Data require an ensemble of different structures Different trail structures only partially Structures 1,6,7,18 explain the scattering data all present in solution Gag summary: Full length Gag is composed of well folded domains with intervening flexible linkers Matrix domain association to the membrane is helped by a covalent lipid modification (N-myrsilation Capsid: a Gag cleavage fragment undergoes local structural change: N- terminus of NCA folds back on itself How does Gag assemble into 2 different (immature and mature) Cryo EM: Structure of assembled immature viral particles EM reconstruction of capsid arrangement in immature form of the viral particle Gag trimer? MA hexamer? CA dimer? hexameric lattice trimer? NC multimer? 19  Structure of assembled mature viral particles (i.e. only capsid) EM reconstruction of model based on crystal structures and capsid arrangement in cryo-EM reconstructions of in-vitro mature form of the viral assembled viral like particles particle Blue N-CA: Forms hexamers Yellow: C-CA forms dimers CA N-CA C-CA 20 Li et al., 2000  Summary: in vitro Gag assemblies “Immature” viral particle – Overall shape is spherical interfaces: – MA trimer; NCA hexamer, CCA dimer – NCA hexamers are only partially ordered “Mature” viral particle (capsid only) – Overall shape is a cone (fullerene cage) Interfaces: – NCA hexamer (some pentamers & some heptamers) – CCA dimers Are these particles generated in vitro representative of the viruses in vivo? Cryo-EM reconstruction of mature viral particles from infected cells Viral Cores obtained from virally infected cells have variable morphology Hence HIV assembly is not homogeneous i.e. interfaces between capsid proteins not unique Inherent flexibility of the interfaces or even domains 23  Can we interfere with assembly? Inhibition of assembly would render virus non-infectious. Target common assembly interface in both mature and immature viral particles CCA domain good candidate – forms dimers in mature and immature viral particles  A phage display selected peptide that inhibits HIV-1 assembly in vitro selected by phage display for binding to capsid (CA) protein: Capsid Assembly Inhibitor (CAI): ITFEDLLDYYGP shown to inhibit HIV-1 assembly in vitro (two step inhibitor): immature-like, spherical particles mature-like, tubular particles DMACANCSP2 DMACANCSP2 CANC CANC + 5x CAI + 1x CAI 200 nm 200 nm Structure of C-CA W184 M185 W184 and M185 critical for assembly of both mature and immature in vitro viral particles Mutation W184A abrogates assembly  NMR chemical shift perturbation of C-CA W184A/M185A by CAI 120 Val221 121 Val181 * Ala204 Lys182 122 Kd=16±1mM 123 124 Ala177  C-CAW184A/M185A without (black), 125 Ala194 with 0.5x (green), 1x (red), 2x (magenta), 4x (cyan) and 7.3x 126 (blue) molar excess of CAI 8.4 8.2 8.0 7.8 7.6 7.4 7.2 1H (ppm) Backbone chemical shift perturbation induced by CAI binding C-CA  all significant shifts occur within amino acids 169 to 191 of C-CA (coloured red), which includes the dimer interface of CA Structural rearrangement of CCA when inhibitor is bound helix2 (179-193) CAI helix1 (161-174) helix4 (211-217)  Plasticity in the interface between C-terminal capsid domain (C-CA) C-CA dimer 900Å2 buried in dimer interface C-CA/CAI dimer 500Å2 buried in dimer interface CAI: 12 residue helical peptide  Summary Reprise of structural methods – Their strength and limitations Case study of HIV viral assembly – Requires full repertoire of different techniques Protein structure and plasticity is an integral factor in HIV assembly – Folded domains – Flexible linkers – Structural plasticity in interfaces  Library Project: Portrait of a Protein Consult Blackboard/Biol2012 Assignments tab: Choose protein from the list Write a review (5 pages) including at least three figures you made yourself (with chimera) using the atomic coordinates of the protein Chimera help desk (if you need it) Tue 5-6pm Deadline Choose Fri 28th March noon protein Download template from blackboard and adhere to the formatting provided there  Library Project: Portrait of a Protein Common questions arising: – Proteins have multiple PDB entries. Multiple organisms, different molecular states, mutations, ligand complexes Use as appropriate that demonstrate the points you want to make. – The available structures do not cover the whole protein. Hence understanding of structure function is incomplete. Future work. – You may include additional diagrams other than the three structure images, include reference if you did not make those yourself. – Referencing: no www addresses! Use Harvard/Vancouver, or journal style references  Portrait of a Protein Questions you may consider: What is the function of the protein in question? How does the structure inform you about the function? Have you described the structure? Does the protein require any processing to reach its final, functional form? Is it functional as a single polypeptide, or is it a dimer, trimer etc.? Is it a singular gene product or are there multiple isoforms of the protein? Is its function/activity regulated at the protein and/or gene level? * Can specific residues or precise structural regions be mapped to specific aspects of its function or regulation? Are there any mutations (disease-related or engineered in) that shed light on the role of specific residues?  Figure legends (forward feedback) Describe everything that is visible on the figure Not good Figure x: Structure of HIV Capsid Good Figure x: Ribbon diagram of the C- and N- terminal domains of HIV capsid labeled respectively NTD in green colour and CTD in teal colour. Regular helices are labelled H1-H11 starting from the N- to C-terminus as well as a 310 helix at the beginning of the C-terminal domain. The N-terminal hairpin loop is coloured yellow and the location of cyclophilin-binding loop is labelled. Dots are used to indicate residues whose structure are not well determined including the labelled linker region between the domains. The figure was created with UCSF chimera (ref) using the PDB entries xyz and abc for the domains respectively and modelled into the EM density of a mature capsid.  What makes a good figure? (forward feedback) Maximize information content – and omit everything that is not relevant to what you are trying to show or say. (omit distractions) Be detailed and specific. – Highlight important features, residues, distances , etc.. add appropriate labels ( e.g. in powerpoint) The text needs to refer to the figures otherwise they are not justified.