Protein structure and alignment

1
Protein structure and alignment

• When you see a protein sequence alignment,
  notice the blocks with higher and lower
  similarity (they are almost always there).
• (most of the time) These are not simply
  stochastic variation they represent regions
  under more or less strong purifying selection.
• These blocks can vary from rather small segments
  to rather long domains (or both).
• Longer blocks usually correspond to different
  protein domains (which can vary as a unit in
  selective pressure).
• Shorter blocks usually correspond to
  intra-domain structural features.

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Kinases form a complex, diverse family
Example from a particular enzyme
(many subtypes)
(many subtypes)
3
CaM Kinase I and CaM Kinase II (CaMKII) (CaM
stands for calcium-calmodulin)

• Very similar in the kinase and calmodulin
  regulatory domains.
• CaMKI is monomeric, whereas CaMKII is a 10-12
  subunit multimer.
• CaMKII most likely arose (a very long time ago)
  after the CaM Kinase domain by fusing a multimer
  formation domain to the C-terminus.

4
CaM Kinase II structure
N
C
multimer
serine-threonine
calmodulin
formation
protein kinase
regulation
12 subunits
with the catalytic
domains facing out
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insect, nematode, chordate CaMKII
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unc-43 --------------------MQLQQINSGAFSVV
RRCVHKTTGLEFAAKIINTKKLSARD rCaMKII
-------MATITCTRFTEEYQLFEELGKGAFSVVRRCVKVLAGQEYPAKI
INTKKLSARD hCaMKI MLGAVEGPRWKQAEDIRDIYDFR
DVLGTGAFSEVILAEDKRTQKLVAIKCIAKEALEGKE rCaMKI
MPGAVEGPRWKQAEDIRDIYDFRDVLGTGAFSEVILAEDKRTQKLV
AIKCIAKKALEGKE
.. . .
..   unc-43 FQKLEREARICRKLQHPNIVRLHDSIQEE
SFHYLVFDLVTGGELFEDIVAREFYSEADAS rCaMKII
HQKLEREARICRLLKHPNIVRLHDSISEEGHHYLIFDLVTGGELFEDIVA
REYYSEADAS hCaMKI GS-MENEIAVLHKIKHPNIVALD
DIYESGGHLYLIMQLVSGGELFDRIVEKGFYTERDAS rCaMKI
GS-MENEIAVLHKIKHPNIVALDDIYESGGHLYLIMQLVSGGELFD
RIVEKGFYTERDAS . . .
.. . .. . ..
  unc-43 HCIQQILESIAYCHSNGIVHRDLKPENL
LLASKAKGAAVKLADFGLAIEVN-DSEAWHGF rCaMKII
HCIQQILEAVLHCHQMGVVHRDLKPENLLLASKLKGAAVKLADFGLAIEV
EGEQQRWFGF hCaMKI RLIFQVLDAVKYLHDLGIVHRDL
KPENLLYYSLDEDSKIMISDFGLSKMED-PGSVLSTA rCaMKI
RLIFQVLDAVKYLHDLGIVHRDLKPENLLYYSLDEDSKIMISDFGL
SKMED-PGSVLSTA . ....
. . . ...
  unc-43 AGTPGYLSPEVLKKDPYSKPVDIWACGVILY
ILLVGYPPFWDEDQHRLYAQIKAGAYDYP rCaMKII
AGTPGYLSPEVLRKDPYGKPVDLWACGVILYILLVGYPPFWDEDQHRLYQ
QIKARAYDFP hCaMKI CGTPGYVAPEVLAQKPYSKAVDC
WSIGVIAYILLCGYPPFYDENDAKLFEQILKAEYEFD rCaMKI
CGTPGYVAPEVLAQKPYSKAVDCWSIGVIAYILLCGYPPFYDENDA
KLFEQILKAEYEFD ... .
. .. ..
  unc-43 SPEWDTVTPEAKSLIDSMLTVNPKKRITADQ
ALKVPWICNRERVASAIHRQDTVDCLKKF rCaMKII
SPEWDTVTPEAKDLINKMLTINPSKRITAAEALKHPWISHRSTVASCMHR
QETVDCLKKF hCaMKI SPYWDDISDSAKDFIRHLMEKDP
EKRFTCEQALQHPWIAGDTALDKNIH-QSVSEQIKKN rCaMKI
SPYWDDISDSAKDFIRHLMEKDPEKRFTCEQALQHPWIAGDTALDK
NIH-QSVSEQIKKN ..
.. . .. . . . . .
  unc-43 NARRKLKGAILTTMIATRNLSSKRSYRLTLG
AEKLVISMKNIEYWQVLLNKIFATYKIKM rCaMKII
NARRKLKGAILTTMLATRNFSGG---------------------------
--------KS hCaMKI FAKSKWKQAFNATAVVRHMR---
------------------------------------- rCaMKI
FAKSKWKQAFNATAVVRHMR--------------------------
-------------- . . . .

continued
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continued (overlapped)
unc-43 SPEWDTVTPEAKSLIDSMLTVNPKKRITADQALK
VPWICNRERVASAIHRQDTVDCLKKF rCaMKII
SPEWDTVTPEAKDLINKMLTINPSKRITAAEALKHPWISHRSTVASCMHR
QETVDCLKKF hCaMKI SPYWDDISDSAKDFIRHLMEKDP
EKRFTCEQALQHPWIAGDTALDKNIH-QSVSEQIKKN rCaMKI
SPYWDDISDSAKDFIRHLMEKDPEKRFTCEQALQHPWIAGDTALDK
NIH-QSVSEQIKKN ..
.. . .. . . . . .
  unc-43 NARRKLKGAILTTMIATRNLSSKRSYRLTLG
AEKLVISMKNIEYWQVLLNKIFATYKIKM rCaMKII
NARRKLKGAILTTMLATRNFSGG---------------------------
--------KS hCaMKI FAKSKWKQAFNATAVVRHMR---
------------------------------------- rCaMKI
FAKSKWKQAFNATAVVRHMR--------------------------
-------------- . . . .

  unc-43 KQCRNLLNKKEQGPPSTIKESSESS-QTIDD
NDSEKGGGQLKHENTVVRADGATGIVSSS rCaMKII
G--G---NKKNDG----VKESSESTNTTIEDED-----------------
---------- .
.. ..   unc-43
NSSTASKSSSTNLSAQKQDIVRVTQTLLDAISCKDFETYTRLCDTSMTCF
EPEALGNLIE rCaMKII ------------TKVRKQEIIKV
TEQLIEAISNGDFESYTKMCDPGMTAFEPEALGNLVE
.... .. ...
..   unc-43
GIEFHRFYFD--GNRKNQ-VHTTMLNPNVHIIGEDAACVAYVKLTQFLDR
NGEAHTRQSQ rCaMKII GLDFHRFYFENLWSRNSKPVHTT
ILNPHIHLMGDESACIAYIRITQYLDAGGIPRTAQSE
... . .....
..... .   unc-43
ESRVWSKKQGRWVCVHVHRSTQPSTNTTVSEF rCaMKII
ETRVWHRRDGKWQIVHFHRSGAPSVLPH----
. .. . .
(note both inter- and intra-domain differences in
conservation)
blue kinase, orange calmodulin binding, green
multimerization
8
Protein structure basics

• proteins consist mostly of a-helices, b-sheets,
  and turns.
• the a-helices and b-sheets typically form the
  framework of the protein.
• the turns and other atypical structures often
  play important binding and catalytic roles.
• the core of the protein is hydrophobic, whereas
  the surface is usually polar or charged.
• most sharp turns (kinks) have glycine or proline.

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alpha helix
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three-stranded antiparallel b-sheet
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three-stranded antiparallel b-sheet, space filled
12
substrate binding cleft
arm swings away on calmodulin binding
rCaMKII SPEWDTVTPEAKDLINKMLTINPSKRITAAEALK
HPWISHRSTVASCMHRQETVDCLKKF rCaMKI
SPYWDDISDSAKDFIRHLMEKDPEKRFTCEQALQHPWIAGDTALDKNIH-
QSVSEQIKKN 297 ..
.. . . ... . . . . .
   rCaMKII NARRKLKGAILTTMLATRN rCaMKI
FAKSKWKQAFNATAVVRHM 316
. . . . .
 
gold and red - calmodulin binding arm and site
respectively
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sliced half-way through the protein
red - charged blue - polar green - hydrophobic
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rCaMKII HQKLEREARICRLLKHPNIVRLHDSISEEGHHYL
IFDLVTGGELFEDIVAREYYSEADAS rCaMKI
GS-MENEIAVLHKIKHPNIVALDDIYESGGHLYLIMQLVSGGELFDRIVE
KGFYTERDAS 119 . . .
. ... . ..
  rCaMKII HCIQQILEAVLHCHQMGVVHRDLKPENL
LLASKLKGAAVKLADFGLAIEVEGEQQRWFGF rCaMKI
RLIFQVLDAVKYLHDLGIVHRDLKPENLLYYSLDEDSKIMISDFGLSKME
D-PGSVLSTA 178 . ..
. . . ... .
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rCaMKII HCIQQILEAVLHCHQMGVVHRDLKPENLLLASKL
KGAAVKLADFGLAIEVEGEQQRWFGF rCaMKI
RLIFQVLDAVKYLHDLGIVHRDLKPENLLYYSLDEDSKIMISDFGLSKME
D-PGSVLSTA 178 . ..
. . . ... .
    rCaMKII AGTPGYLSPEVLRKDPYGKPVDLWACGVI
LYILLVGYPPFWDEDQHRLYQQIKARAYDFP rCaMKI
CGTPGYVAPEVLAQKPYSKAVDCWSIGVIAYILLCGYPPFYDENDAKLFE
QILKAEYEFD 238 ... .
. .. ... .
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Measuring structural similarity

• Structural similarity can persist after sequence
  similarity has reached noise levels.
• More generally, how do you measure two
  structures for degree of similarity?
• Commonly used approach is root mean square
  deviation (RMSD) between the positions of matched
  backbone atoms.

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No statistically significant sequence similarity
RMSD for shared regions 3.5 Angstroms
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Illustration of three points on a structure of
poorly known function

• gaps in alignments tend to be on surface loops
• areas of highest conservation tend to be at key
  sites (e.g. active sites of enzymes) and in core
  structural elements
• BUT when positive selection acts, binding faces
  may tend to be the parts that vary.

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MATH domain containing genes a mystery family
in C. elegans
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For Thursday Download either Rasmol or Cn3D
structure viewer and a protein structure of your
choice. Send in an image of some view of the
protein with only the backbone showing and an
alpha helix or beta sheet colored differently
from the rest. (Youll have to read documentation
from your viewer to figure out how to do
this.) Final assignment will be posted by this
evening. Due on the last day of exam week.