Myosin V Discrimination Between Distinct Cargoes

Presented as a part of Protein structure course (Comp. Bio.) at IMSc

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1. MYOSIN V DISCRIMINATION BETWEEN DISTINCT CARGOES Vinay Vaibhav The Institute of Mathematical Sciences Sep. 07, 2017 Pashkova, Natasha, et al. "Structural basis for myosin V discrimination between distinct cargoes." The EMBO Journal 25.4 (2006): 693-700.
2. https://www.uni-muenster.de/Cells-in-Motion/research/areaA/index.php Microtubules: Tubulin Actin filaments: Actin The Cell Cytoskeleton
3. Actin-based motility: Myosin — 18 different myosin families — Myosin V : Processive cargo transporter — 38 myosin genes in human genome (3 class V) — 5 myosin genes in yeast (2 class V: MYO2p and MYO4p) Tubulin-based motility: Kinesin and Dynein — Kinesin takes its cargo to + end (away from nucleus) — Dynein takes its cargo to - end Protein molecules which use energy from ATP hydrolysis to move cargo along the cytoskeleton Motor Proteins
4. Four structural domain: Head or motor domain, Neck or lever arm, Tail or rod region and Tail or globular tail domain (GTD) Cellular and Molecular Life Sciences 65.9 (2008): 1378-1389. Myosin V
5. Myosin V Kinesin
6. Myosin V motor: Myo2p (and Myo4p) Known to carry at least 6 different types of cargoes — Attachment / Detachment — Regulation of motor activity Identified organelle-specific receptors in other myosin V proteins Eukaryotic Single-celled Fungus Model organism Yeast: Saccharomyces cerevisiae Modern Electron Microscopy in Physical and Life Sciences. InTech, 2016.
7. Crystal grown at 4oC : Hanging-drop vapour-diffusion Crystal appeared within one week Residues: 1087-1574 Cargo-binding Domain : Two Subdomains — Subdomain I : 1131-1345 — Subdomain II : 1346-1574 Pashkova, N., Catlett, N. L., Novak, J. L., Wu, G., Lu, R., Cohen, R. E., & Weisman, L. S. (2005). Myosin V attachment to cargo requires the tight association of two functional subdomains. The Journal of cell biology, 168(3), 359-364. Subdomains alone: No activity Coexpression of subdomains separately (in vivo): Functional complex Simultaneous expression of subdomains: Function inhibited Crystallization of Myo2p GTD Yeast vacuole Secretory vesicles PDB ID: 2F6H
8. Purple: Residues observed in crystal Residues missing from structure but present in detected polypeptide — Flexibility of polypeptide chain Residues missing from both structure and detected polypeptide — Digested during proteolysis or incubation in crystallisation buffer 1131 STPSSGNNHIDSLSVDRENGVNATQINEELYRLLEDTEILNQEITEGLLKGFEVPDAGVA 1191 IQLSKRDVVYPARILIIVLSEMWRFGLTKQSESFLAQVLTTIQKVVTQLKGNDLIPSGVF 1251 WLANVRELYSFVVFALNSILTEETFKNGMTDEEYKEYVSLVTELKDDFEALSYNIYNIWL 1311 KKLQKQLQKKAINAVVISESLPGFSAGETSGFLNKIFANTEEYTMDDILTFFNSIYWCMK 1371 SFHIENEVFHAVVTTLLNYVDAICFNELIMKRNFLSWKRGLQLNYNVTRLEEWCKTHGLT 1431 DGTECLQHLIQTAKLLQVRKYTIEDIDILRGICYSLTPAQLQKLISQYQVADYESPIPQE 1491 ILRYVADIVKKEAALSSSGNDSKGHEHSSSIFITPETGPFTDPFSLIKTRKFDQVEAYIP 1551 AWLSLPSTKRIVDLVAQQVVQDGH Likely peptides (underlined) that correspond to the peaks from MALDI-TOF MS analysis: Peak 1 – 12154.20 Da Peptide - 12193.52 Da (underlined in dashed black) Peak 2 - 13701.32 Da Peptide - 13700.56 Da (underlined in dashed pink) Amino acid sequence of the Myo2p tail MALDI-TOF Mass Spectrometry Analysis
9. Yellow : Identical residues Green: Residues with strong similarity Cyan: Residues with weak similarity Broken lines: Disordered residues red. Identical residues are shaded in yellow, residues with strong similarity in green and with weak similarity in light blue. MY5A-mouse RSQLVSQKEAIQPKDDKNTMTDSTILLEDVQKMKDKGEIAQAYIGLKETNRLLESQLQSQ Sea-urch RRRQWALQEGDSVETGTNTEQHMLVSDEDLDNLQLDSSLISDYKSVKTTNQILEKEIQAL Drosoph QSQSMRSLEPESLQMRGN----------------DVNELMEAFHSQKLINRQLESELKAI Myo2p ---------------------------------NFNNMMLENSDLSPNDLNLKSRSTPSS 1135 Ustilago ---------------------------------NGYTTGGSKRRPRRHSEAGPWSDVPAG HUM-2 REEAVELRSMLSSHFEKQSVAGSSGYRRSPRPDSGHCSGADSEDGSSGADLEEDLCIERQ . H1 H1 MY5A-mouse KRSHENEAEALRGEIQSLKEENNRQQQLLAQNLQLPPEARIEASLQHEITRLTNENLYFE Sea-urch RMNYDYEKTDLKEQIGKLQQDNERQQSIIGENLKLTPAARVSQAIQFELSRLTNENINLM Drosoph TEEHNSKLVEMTQEIERLNNEKDELQKVMFESIDEFEDS--------NVDTLRQNDRYLR Myo2p GNNHIDSLSVDRENGVNATQIN------------------------------EELYRLLE 1165 Ustilago RDEHEEAMMAAKRSAATANRHVSVAFGLEGHQIPGFGQRNGYDDEYDQDDPSEEIIRILE HUM-2 CRHLKNLAENLTKMLTNQNLEIERLQQQLRFSESQTVFRPSDCSLDEAVRGAHKQTQLLA H2 MY5A-mouse ELYADDPKKYQSYRISLYKRMIDLMEQLEKQDKTVRKLKKQLKVFAKKIGELEVGQMENI Sea-urch EEKEYLEKFVKKLKKQLKAAHKRMQSTSSADLGGVATVNNIPDVHLSGVPSEFNDTPESS Drosoph RELQKAVAQFLLVQEELKLANAKLKAYRQDGGQLEHKIEEEMIRNKSNGTSADVGANVTK Myo2p DTEILNQEITEGLLKGFEVPDAGVAIQLSKRD---------------------------- Ustilago NEEQLDEDVLNGLIRYLKVPAPSLQNPPSPKE---------------------------- HUM-2 QQNMDLNDKLTRQSEELAEARAQLRGYSGPLGLENASDEEIIRLEAFEKGSIKHSGFLEV H3 MY5A-mouse SPGQIIDEPIRPVNIPRKEKDFQGMLEYKREDEQKLVKNLILELKPRGVAVNLIPGLPAY Sea-urch QANVRVKE----------REEMMGMLEYKAEDEPKLLKMVIIDFIPEAAEG-HLPGLPAY Sequence Alignment of the Myosin V GTD
10. Myo2p GNNHIDSLSVDRENGVNATQIN------------------------------EELYRLLE 1165 Ustilago RDEHEEAMMAAKRSAATANRHVSVAFGLEGHQIPGFGQRNGYDDEYDQDDPSEEIIRILE HUM-2 CRHLKNLAENLTKMLTNQNLEIERLQQQLRFSESQTVFRPSDCSLDEAVRGAHKQTQLLA H2 MY5A-mouse ELYADDPKKYQSYRISLYKRMIDLMEQLEKQDKTVRKLKKQLKVFAKKIGELEVGQMENI Sea-urch EEKEYLEKFVKKLKKQLKAAHKRMQSTSSADLGGVATVNNIPDVHLSGVPSEFNDTPESS Drosoph RELQKAVAQFLLVQEELKLANAKLKAYRQDGGQLEHKIEEEMIRNKSNGTSADVGANVTK Myo2p DTEILNQEITEGLLKGFEVPDAGVAIQLSKRD---------------------------- Ustilago NEEQLDEDVLNGLIRYLKVPAPSLQNPPSPKE---------------------------- HUM-2 QQNMDLNDKLTRQSEELAEARAQLRGYSGPLGLENASDEEIIRLEAFEKGSIKHSGFLEV H3 MY5A-mouse SPGQIIDEPIRPVNIPRKEKDFQGMLEYKREDEQKLVKNLILELKPRGVAVNLIPGLPAY Sea-urch QANVRVKE----------REEMMGMLEYKAEDEPKLLKMVIIDFIPEAAEG-HLPGLPAY Drosoph QKS----------------QNPQGLMKFHSSDLDKILQRLLSALTPRTVVG-LLPGFPAY Myo2p ------------------------------------------------------VVYPAR 1203 Ustilago ------------------------------------------------------VLFPAH HUM-2 YNVPEFAR-------------------------------IIVCELKPTLARLLTKNLPAY H3 H4 H5 MY5A-mouse ILFMCVRHADYLNDDQKVRSLLTSTINSIKKVLKKRG--DDFETVSFWLSNTCRFLHCLK Sea-urch IIFMCIRHADFVNDDRKVKALLTGVINGIKKTVKKHF--EDFEYVSFWLTNATRLLHNLK Drosoph LIFMCIRYTDLTNADDDVRELLSKFVIQIKKMHRTP---HPIENRVIWLVNSITLLNLMK Myo2p ILIIVLSEMWRFGLTKQSESFLAQVLTTIQKVVTQLKGNDLIPSGVFWLANVRELYSFVV 1263 Ustilago LISLVTNEMWKYGLVRESERFLANVMQTIQQHVMSFQGEDAIIPGIFWLSNVHEILSFVC HUM-2 LLVAAFRNHDEKRDETALTGLFSSVHLVLKDTISRS---HDLDLLSLWLVNLWRLFNLLR H5 H6
11. MY5A-mouse QYSGEEGFM----KHNTSRQNEHCLTNFDLAEYRQVLSDLAIQIYQQLVR-VLENILQPM Sea-urch QYSGEESFS----SKNTERQNEHCLRNFDLSEYRHVMNDLGIHIYQMLIR-IIENSVQPM Drosoph QYGDVDEYV----KFNTEKQNQQQLKNFNLFEYRRVILDLIVNLYQALIM-QIQGLLDPK Myo2p FALNSILT--EETFKNGMTDEEYKEYVSLVTELKDDFEALSYNIYNIWLK-KLQKQLQKK 1320 Ustilago IAESDMLQGIGPGVDGAARSFEWGDYERLVTIVKHDLDSLEYNIYHTWMQ-EAKKRLHKM HUM-2 QYSGEDSQP-EWHVANTETQNSYRFKAYDVAPIRDQLKLRIEECYTSLMKKAIEHVLSPK H6 H7 MY5A-mouse IVSGMLEHETIQGVSGVKPTGLRKRTS--SIAD--EGTYTLDSILRQLNSFHSVMCQHGM Sea-urch IVTAMLEGEMAGLVS-SKPTGVRGSNS--TIREREVKDVSIDSLIKQLGTYITVMNVHGM Drosoph IVPAILNNDEIQRGRQAHGMRSRATSIGASSSPEHGGGPAWKQLIGQLEHFYKQFQHFGL Myo2p AINAVVISESLPGFSAGETSG----FLNKIFAN--TEEYTMDDILTFFNSIYWCMKSFHI 1374 Ustilago VIPALVESQSLPGFVTSDHSGR---LFNRLLSNNSTPMHTMDDILGILNKVWKSLKSYYV HUM-2 IVPGILQHESSSDLMTAGQERR-----DRNSGSVESQRKSLDDLLQFMEIVHTKLTTYGG H8 H9 MY5A-mouse DPELIKQVVKQMFYIVGAITLNNLLLRKDMCSWSKGMQIRYNVSQLEEWLRDKNLMNSGA Sea-urch DPELVKQVARQALYLITASTINNILLRKDMCHWSKGVQIRYNLSELEEWLRSSRLYDKMM Drosoph DNCYAEQIFHQLLYFICAVALNCLMLRGDICMWETGMIIRYNIGCIEDWVRSKKMSN-DV Myo2p ENEVFHAVVTTLLNYVDAICFNELIMKRNFLSWKRGLQLNYNVTRLEEWCKT--HGLTDG 1432 Ustilago EPSVTQQVVTELLKLIGVTSFNDLLMRRNFCSWKRAMQIQYNITRIEEWCKS--HDMPEG HUM-2 DDIVVKQVIGQMARWMCALALNYMMFRRELCNFEKAIQIKHNVTQIQNWLNAK--GLSDC H10 H11 H12 H13 MY5A-mouse KETLEPLIQAAQLLQVKKKTDDDAEAICSMCNALTTAQIVKVLNLYTPVNEFEERVSVSF Sea-urch ETTLEPLVQVAQLLQVKKRTDDDVGIICDTCTQLTVTQIIKILNLYTPD-EYEKRTEIAF H5 H6
12. Drosoph DNCYAEQIFHQLLYFICAVALNCLMLRGDICMWETGMIIRYNIGCIEDWVRSKKMSN-DV Myo2p ENEVFHAVVTTLLNYVDAICFNELIMKRNFLSWKRGLQLNYNVTRLEEWCKT--HGLTDG 1432 Ustilago EPSVTQQVVTELLKLIGVTSFNDLLMRRNFCSWKRAMQIQYNITRIEEWCKS--HDMPEG HUM-2 DDIVVKQVIGQMARWMCALALNYMMFRRELCNFEKAIQIKHNVTQIQNWLNAK--GLSDC H10 H11 H12 H13 MY5A-mouse KETLEPLIQAAQLLQVKKKTDDDAEAICSMCNALTTAQIVKVLNLYTPVNEFEERVSVSF Sea-urch ETTLEPLVQVAQLLQVKKRTDDDVGIICDTCTQLTVTQIIKILNLYTPD-EYEKRTEIAF Drosoph LTALAPLNQVSQLLQSRK-SEQDVQTICDLCTSLSTAQVLKVMKSYKLDDYESEITNVFL Myo2p TECLQHLIQTAKLLQVRKYTIEDIDILRGICYSLTPAQLQKLISQYQVADYESPIPQEIL 1492 Ustilago TLQLEHLMQATKLLQLKKATLGDIDIIYDVCWMLTPTQIQKLISHYYVADYENPISPEIL HUM-2 RDHFEPLVQACHLLQSRKDPSNLDTLCGEMTSRLKPRQVVAILQHYDPSDEMEDGLSPEF H13 H14 MY5A-mouse IRTIQMRLRD----------------------RKD---SPQLLMDAKHIFPVTFPFNPSS Sea-urch IRKVQSRLAN----------------------RNDPKRESQLLIDAKHTFPVTFPY---- Drosoph EKLTEKLNAR----------------------QMQKSNSDEFTIDQKFIQPFKVVFRYSD Myo2p RYVADIVKKEAALSSSGNDSKGHEHSSSIFITPETGPFTDPFSLIKTRKFDQVEAYIPAW 1552 Ustilago KAVASRVVPNDRND--------------HLLLPPEVDEAGPYELPLPREVTGIETYCPAY HUM-2 LVQIQKKLNERAIAN----------------NDPIEDKDKLIMLGTYLPPFDTQPFSYSD H15 MY5A-mouse LALETIQIPASLGLGFIARV-- Sea-urch ---------------------- Drosoph IKLEDIELPSHLNLDEFLTKI- Myo2p LSLPSTKRIVDLVAQQVVQDGH 1574 Ustilago ISVPAIRRLASRVA-------- HUM-2 FPLETLSLPSCLHMQSVCRLV-
13. 15 amphipathic alpha-helices 2 five-helical bundles: H2 to first half of H6 and distal half of H6 to H10 H6 (45 residues): Connects the two bundles Two helical bundles correspond to subdomain I and II Pashkova, Natasha, et al. "Structural basis for myosin V discrimination between distinct cargoes." The EMBO Journal 25.4 (2006): 693-700. Subdomain 1 Subdomain 1I Structure
14. 90o offset Subdomain I Subdomain I : 1131-1345 Subdomain II : 1346-1574 Subdomain I : 1131-1309 and 1528-1574 Subdomain II : 1310-1527 Why coexpression require both subdomains? - Interaction between H5 and H7 - Extended C-terminal loop
15. Vacuole-binding Region Identified seven point mutants - Inhibit binding of vacuoles not vesicles - Vac17p: Vacuole-specific receptor complex D1297N/G, L1301P, N1304S/D N1307D 6 mutations cluster to four residues in an 11 amino acid span Lie along alpha helix H6 H4: Mutation of Gln1233 to Arg, affects vacuole movement Vacuole-binding region: Subdomain 1 The EMBO Journal 25.4 (2006): 693-700.
16. Secretory Vesicle-binding Region Identified the areas of high sequence conservation Presence of several large patches of highly conserved residues Orange: Bind to secretory vesicles (not conserved with Myo4p) Blue: Subdomain I Red: Subdomain II Cyan: Vacuole-binding region
17. When motor is turned off The Inhibited State Cellular and Molecular Life Sciences 65.9 (2008): 1378-1389.
18. Myosin V From Yeast and Humans PloS one 8.12 (2013): e82065.
19. Thank You