I.  Djagaeva  et  al.  2  SI  

Figure S1.

10 20 30 40 50 Human Kif5A ---MAETNNECSIKVLCRFRPLNQAEILRGDKFIPIFQGD---DSVVIGGKPYVFDRVFP

: : ::::.:::::::..: :.::. : .. . . :.:: :.::.:: Fly Khc MSAEREIPAEDSIKVVCRFRPLNDSEEKAGSKFVVKFPNNVEENCISIAGKVYLFDKVFK

10 20 30 40 50 60 β1 L1 β1a β1b β1c B2

60 C 70 N 80 90 100 110 Human Kif5A PNTTQEQVYHACAMQIVKDVLAGYNGTIFAYGQTSSGKTHTMEGKLHDPQLMGIIPRIAR

::..::.::. : .:: :::::::::::::::::::::::::: . : .::::::. Fly Khc PNASQEKVYNEAAKSIVTDVLAGYNGTIFAYGQTSSGKTHTMEGVIGDSVKQGIIPRIVN 70 N74 90 100 110 L68

L3 α1 β3 L4 α2 L5

120 130 140 150 160 170 Human Kif5A DIFNHIYSMDENLEFHIKVSYFEIYLDKIRDLLDVTKTNLSVHEDKNRVPFVKGCTERFV

:::::::.:. ::::::::::.:::.:::::::::.:.::::::::::::.::: ::::: Fly Khc DIFNHIYAMEVNLEFHIKVSYYEIYMDKIRDLLDVSKVNLSVHEDKNRVPYVKGATERFV

Q32 140 150 K23 K19 α2 L6 β4 L7 β5 L8 β5a β5b

W

180 190 T NCQ 210 220 L Human Kif5A SSPEEILDVIDEGKSNRHVAVTNMNEHSSRSHSIFLINIKQENMETEQKLSGKLYLVDLA ::::....::.:::::::.::::::::::::::.::::.::::.:...::::::::::::

Fly Khc SSPEDVFEVIEEGKSNRHIAVTNMNEHSSRSHSVFLINVKQENLENQKKLSGKLYLVDLA 190 200 210 220 230 T61

α3 L9 α3a β6 L10 β7 V66

H Del L

E 240 K N SN 260 270 C C 290 Human Kif5A GSEKVSKTGAEGAVLDEAKNINKSLSALGNVISALAEGTKSYVPYRDSKMTRILQDSLGG ::::::::::::.:::::::::::::::::::::::.:.:...::::::.:::::.::::

Fly Khc GSEKVSKTGAEGTVLDEAKNINKSLSALGNVISALADGNKTHIPYRDSKLTRILQESLGG F17 K2 F75 260 270 N37 4M 300

L11 I62 α4-ext α4 L12 α5

E63

300 310 320 330 340 350

Human Kif5A NCRTTMFICCSPSSYNDAETKSTLMFGQRAKTIKNTASVNLELTAEQWKKKYEKEKEKTK : :::. :::::.:.:..:::::: ::.::::.::.. :: :::::.::..:::::::. Fly Khc NARTTIVICCSPASFNESETKSTLDFGRRAKTVKNVVCVNEELTAEEWKRRYEKEKEKNA

C36 310 S10a V67 340 350 K10b β8 L14 α6 S64 α7

Neck linker

V 370 380 390 Human Kif5A AQKETIAKLEAELSRWRNGENV----------------PETERLAGEEAALGAELCEETP

: . ::: ::.::: ::.: :. : :.. :: : : . Fly Khc RLKGKVEKLEIELARWRAGETVKAEEQINMEDLMEASTPNLEVEAAQTAAAEAALAAQRT 370 380 K11 390 400 410 420

Hinge 1

                                                         

I.  Djagaeva  et  al.   3  SI  

400 410 420 430 440 450 Human Kif5A VNDNSSIVVRIAPEERQKYEEEIRRLYKQLDDKDDEINQQSQLIEKLKQQMLDQEELLVS

. : : . .: .:. . : .:::.::::::.::::::: :.::.:...::::... Fly Khc ALA--NMSASVAVNEQARLATECERLYQQLDDKDEEINQQSQYAEQLKEQVMEQEELIAN

430 440 450 460 470 Stalk Coil 1

460 470 480 490 500 510 Human Kif5A TRGDNEKVQRELSHLQSENDAAKDEVKEVLQALEELAVNYDQKSQEVEEKSQQNQLLVDE

.: . : .: :....:.::..::.::::::::::::::::::::::...:... . : .: Fly Khc ARREYETLQSEMARIQQENESAKEEVKEVLQALEELTVNYDQKSQEIDNKNKDIDALNEE 480 490 I58 K34 520 530

520 530 540 550 560 570

Human Kif5A LSQKVATMLSLESELQRLQEVSGHQRKRIAEVLNGLMKDLSEFSVIVGNGEIKLPVEISG :.:: ... . .:::.:...:.::.:::.:.:..:..::.: . .. :: .. ...:. Fly Khc LQQKQSVFNAASTELQQLKDMSSHQKKRITEMLTNLLRDLGEVGQAIAPGESSIDLKMSA

540 550 560 570 580 590 Hinge 2

580 590 600 610 620 630 Human Kif5A -------AIEEEFTVARLYISKIKSEVKSVVKRCRQLENLQVECHRKMEVTGRELSSCQL

.::.::.:::.:::.:.:.:....:: ..:. :.. ..:. ..:. .: Fly Khc LAGTDASKVEEDFTMARLFISKMKTEAKNIAQRCSNMETQQADSNKKISEYEKDLGEYRL

600 610 620 630 640 650 Stalk Coil 2

640 650 660 670 680 690 Human Kif5A LISQHEAKIRSLTEYMQSVELKKRHLEESYDSLSDELAKLQAQETVHEVALKDKEPDTQD

:::::::...:: : :. .: ::: :::. ::: .: :::.: : : : ..: : Fly Khc LISQHEARMKSLQESMREAENKKRTLEEQIDSLREECAKLKAAEHVSAVNAEEK----QR 660 670 680 690 700 710

700 710 720 730 740 750

Human Kif5A ADEVKKALELQMESHREAHHRQLARLRDEINEKQKTIDELKDLNQKLQLELEKLQADYEK :.:... .. ::. :::: ::...::::: ::. .::.::..::: : ... ::::: Fly Khc AEELRSMFDSQMDELREAHTRQVSELRDEIAAKQHEMDEMKDVHQKLLLAHQQMTADYEK

720 730 Q6 750 760 770

K 760 770 780 790 800 810 Human Kif5A LKSEEHEKSTKLQELTFLYERHEQSKQDLKGLEETVARELQTLHNLRKLFVQDVTTRVKK ...:. :::..::.. . ::.::...::::::.:::.:::::::::::::::. :..:

Fly Khc VRQEDAEKSSELQNIILTNERREQARKDLKGLEDTVAKELQTLHNLRKLFVQDLQQRIRK 780 790 800 810 820 830

Stalk Coil 3 820 830 840 850 860 870

Human Kif5A SA-EMEPEDSGGIHSQKQKISFLENNLEQLTKVHKQLVRDNADLRCELPKLEKRLRATAE .. . : :..:: .::::::::::::.:::::::::::::::::::::::::::: : :

Fly Khc NVVNEESEEDGGSLAQKQKISFLENNLDQLTKVHKQLVRDNADLRCELPKLEKRLRCTME 840 850 860 870 880 890 Stalk Coil 4

880 890 900 910 920

Human Kif5A RVKALEGALKEAKEGAMKDKRRYQQEVDRIKEAVRYKSSGKRGHSAQIAKPVRPGHYP-A :::::: ::::::::::.:..::: :::::::::: : :.:: .::::::.: :. : Fly Khc RVKALETALKEAKEGAMRDRKRYQYEVDRIKEAVRQKHLGRRGPQAQIAKPIRSGQGAIA

900 910 K76 930 T15 S22 Globular Tail IAK F77

930 940 Human Kif5A SSPTNPYGTRSPEC-ISYTNS

. : :: .. .:: Fly Khc IRGGGAVGGPSPLAQVNPVNS

960 970

I.  Djagaeva  et  al.  4  SI  

Figure  S1      Comparison  of  human  Kif5A  and  Drosophila  Khc  sequences,  secondary  structures,  and  mutant  alleles.  The  positions  of  Drosophila  Khc  and  of  human  Kif5A  mutant  amino  acid  changes  are  noted  relative  to  known  structural  regions  of  Khc.  Human  Kif5A  mutant  amino  acid  changes  are  above  the  sequence  and  are  highlighted  blue  (BLAIR  et  al.  2006;  CRIMELLA  et  al.  2011;  FICHERA  et  al.  2004;  GOIZET  et  al.  2009;  LO  GIUDICE  et  al.  2006;  MUSUMECI  et  al.  ;  REID  et  al.  2002;  SCHULE  et  al.  2008;  TESSA  et  al.  2008).  Drosophila  Khc  missense  changes  are  noted  below  the  sequence  and  are  highlighted  yellow  with  corresponding  Khc  allele  numbers  in  red.  For  the  Khc  head,  a-­‐helix  forming  residues  are  purple,  b-­‐strands  are  green,  and  intervening  loops  are  not  highlighted.  Portions  of  some  loops  known  to  have  special  functions  are  underlined  and  named  below  the  sequence.  For  the  stalk  and  tail,  coiled-­‐coil  forming  α-­‐  helical  regions  are  noted,  as  are  flexible  hinge  regions  (HACKNEY  2007;  KULL  et  al.  1996;  MORII  et  al.  1997;  VALE  and  FLETTERICK  1997).  Note  that  the  boundaries  of  some  individual  structures  are  a  bit  ambiguous  because  of  slight  differences  in  amino  acid  sequence  numbering  from  different  references.  For  this  Figure,  the  SwissProt  data  base  accession  number  for  Kif5A  is  Q12840  and  for  Drosophila  melangaster  Khc  is  P17210.  Parameters  of  the  sequence  alignment  used  were:  matrix  file  BLOSUM50,  gap  open/ext:  -­‐12/-­‐2.        

I.  Djagaeva  et  al.   5  SI  

Figure S2. Predicted Drosophila Khc structural changes caused by the HSP-like Khc74

D79N (human D73N) mutation. A) In wild-type, D79 (yellow) should form a hydrogen

bond with R303 (cyan) as noted by the double ended arrow and 2.8Å spacing. D8

(green) adjoining the amino-terminal (N-term) coverstrand is free to rotate from the

depicted ADP bound state to an ATP-bound state as implied by the curved arrow. In all

panels orange shows visible parts of α-Helix 4 and Loop 11. B) A space-filling

rendering of wild type to illustrate docking of the neck linker (red ribbon) with the head

during the Khc force-generating conformation change triggered by ATP binding. C)

Modeling of the Khc74 D79N mutation suggests that lack of hydrogen bonding between

                                                                           Figure  S2      Predicted  Drosophila  Khc  structural  changes  caused  by  the  HSP-­‐like  Khc74  D79N  (human  D73N)  mutation.  A)  In  wild-­‐type,  D79  (yellow)  should  form  a  hydrogen  bond  with  R303  (cyan)  as  noted  by  the  double  ended  arrow  and  2.8Å  spacing.  D8  (green)  adjoining  the  amino-­‐terminal  (N-­‐term)  coverstrand  is  free  to  rotate  from  the  depicted  ADP  bound  state  to  an  ATP-­‐bound  state  as  implied  by  the  curved  arrow.  In  all  panels  orange  shows  visible  parts  of  α-­‐Helix  4  and  Loop  11.  B)  A  space-­‐filling  rendering  of  wild  type  to  illustrate  docking  of  the  neck  linker  (red  ribbon)  with  the  head  during  the  Khc  force-­‐generating  conformation  change  triggered  by  ATP  binding.  C)  Modeling  of  the  Khc74  D79N  mutation  suggests  that  lack  of  hydrogen  bonding  between  N79  and  R303  allows  a  new  bond  between  R303  and  D8,  as  noted  by  the  double-­‐ended  arrow  and  3.5Å  spacing.  That  bond  is  predicted  to  resist  the  ATP-­‐induced  rotation  of  N8.  D)  A  space-­‐filling  rendering  of  the  N79  mutant  protein  in  an  ATP  bound  state.  Note  that  the  N-­‐terminal  strand  is  positioned  to  block  neck  linker  docking.  More  subtle  changes  can  be  seen  in  the  structures  of  Loop11  and  α-­‐Helix  4  in  the  mutant.    These  models  were  developed  using  a  Monte  Carlo  algorithm  with  Rosetta  starting  with  coordinates  for  the  Drosophila  Khc  head  from  Kaan  et  al.  (KAAN  et  al.  2011).  Each  model  represents  the  most  common  low  energy  state  generated  by  5000  different  tertiary  structure  predictions.  Final  rendering  of  all  structures  was  done  using  Pymol.  On  amino  acid  side-­‐chains,  red  indicates  oxygen  and  dark  blue  indicates  nitrogen.