Structure Prediction and Modeling of a Eukaryotic Member of the Major Facilitator Superfamily

1
Structure Prediction and Modeling of a Eukaryotic
Member of the Major Facilitator Superfamily

• Gaurav Narale

2
Major Facilitator Superfamily (MFS)

• MEMBRANE TRANSPORT
• Largest secondary transporter protein family
  known so far with more than 1000 members
  identified.1
• Use a solute gradient to drive the translocation
  of substrates such as ions, sugars, amino acids,
  peptides and other hydrophilic solutes.2
• Typically 400-600 amino acids long.
• 12 transmembrane ?-helices, with both the N- and
  C-termini in the cytosol.3
• Two six-helix halves connected by a central loop.
• Found in all three kingdoms of living organisms.

3
Identifying Templates and Targets

• TEMPLATES - Two known structures
• Lactose Permease (LacY) E. Coli
• Glycerol-3-Phosphate Transporter (GlpT) E. Coli
• Sequence identity between the two is negligible
  (9).
• CE algorithm for structural alignment indicates
  that they superimpose over most of their chain
  length (RMSD3.7Å)
• 1st GOAL To find a Eukaryotic member of the MFS
  that shows enough sequence identity with one of
  the known structures to allow reasonable
  alignment.

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Function and Mechanism of LacY and GlpT
Both use a solute gradient to drive translocation
of substrate - LacY mediates the coupled
transport of lactose and H - GlpT catalyzes the
exhange of glycerol-3-phosphate for phosphate

• Alternating-Access Model
• Outward-facing conformation exposed to the
  extracellular side.
• Inward-facing conformation exposed to the
  cytoplasm.
• Ribbon Representation
• Amino-terminal domain (blue).
• Carboxyl-terminal domain (green).
• Bends and other irregularities in the ?-helices
  are indicated by deviations from ideally straight
  and continuous helical ribbon.

5
Identifying Templates and Targets

• Lactose Permease (LacY)
• Obtained protein pdb file from protein data bank
  (1PV6) and extracted amino acid sequence in FASTA
  format. www.rcsb.org/pdb
• Searched for a TARGET with high sequence identity
  using NCBI BLAST. www.ncbi.hlm.nih.gov
• General search against all organisms 2
  iterations, threshold 0.005
• - hits were mainly bacterial proteins.
• 2. Saved the results as a profile (PSSM)
• 3. More sensitive search using the original
  sequence as well as the saved profile as input
  while limiting to a eukaryotic search 2
  iterations, threshold 0.01
• Unable to identify a suitable target.

6
Identifying Templates and Targets

• Glucose-3-Phosphate Transporter (GlpT)
• Obtained protein pdb file from protein data bank
  (1PW4) and extracted amino acid sequence in FASTA
  format. www.rcsb.org/pdb
• Searched for a TARGET with high sequence identity
  using NCBI BLAST. www.ncbi.hlm.nih.gov
• General search against all organisms 2
  iterations, threshold 0.005
• Obtained a suitable TARGET Glucose-6-Phosphate
  Translocase
• Homo Sapien
• 3. Utilized BLink to identify several eukaryotic
  close targets for use in multiple sequence
  alignments.

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Multiple sequence alignment

• Only template and target - initial review
• Both templates, target and close targets
• 15 proteins similar to the target selected from
  different species to get a better alignment
• Only template and target extracted
• Around 30 similarity between template and
  target
• Well distributed alignment

8
Alignment using FUGUE
10 20 30 40
50 hs1pw4a ( 5 ) fkpaphkarlpaaeidptYrrl
rwqIflGIffGyaAYylVRkNFALAMpy QUERY g6pt
-------------MAAQGYGYYRTVIFSAMFGGYSLYYFNRKTFSFVM
PS
aaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaa
60 70 80
90 100 hs1pw4a ( 55 )
L-veqgfsrgDLGfALSGISiAygfSkfimgsvSdrsnPrvfLPaGLilA
QUERY g6pt LVEEIPLDKDDLGFITSSQSAAYAIS
KFVSGVLSDQMSARWLFSSGLLLV
aaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaa
110 120
130 140 150 hs1pw4a ( 104 )
AavMlfMGfvpwATssiavMfvlLflCGwfQGmGwpPCgrTmvhwwsqke
QUERY g6pt GLVNIFFAWSSTV----PVFAALWFL
NGLAQGLGWPPCGKVLRKWFEPSQ
aaaaaaaaa aaaa aaaaaaaaaaaaaaa aaaaaaaaa
a 160 170
180 190 200 hs1pw4a ( 154 )
rggivsVwncAhNvggGiPPllFllGmawfndwhAALYmPAfcAilvA
lf QUERY g6pt FGTWWAILSTSMNLAGGLGPILAT
ILAQSY-SWRSTLALSGALCVVVSFL
aaaaaaaaaaaaaaaa aaaaaaaaaaa
aaaaaaaaaaaaa 210
220 230 240 250 hs1pw4a
( 204 ) AfamMrdTpqsCglppiee-----ykndtakqifmq
yVlpnklLwyIAiA QUERY g6pt
CLLLIHNEPADVGLRNLDPMPSEGKKGSLKEESTLQELLLSPYLWVLSTG
aaaa
aaaaaa aaaaaaaaa
260 270 280 290
300 hs1pw4a ( 262 ) NvfVyLLRYGiLDwSPtylkev
KhfaldkSSwAYflYEyagipGTllCgw QUERY g6pt
YLVVFGVKTCCTDWGQFFLIQEKGQSALVGSSYMSALEVGGLVGSIAA
GY aaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaa
310 320 330 340
350 hs1pw4a ( 312 ) msdkv----------frgnrGa
TGvfFMtlVtiaTivywmnpagNptvdm QUERY g6pt
LSDRAMAKAGLSNYGNPRHGLLLFMMAGMTVSMYLFRVTVTSDSPKLW
IL aaaa
aaaaaaaaaaaaaaaaaa aaaaa
360 370 380 390
400 hs1pw4a ( 352 )
iCmivIGflIyGPvmLIglHAleLApkkAagtAagfTglfGylgGSvaAs
QUERY g6pt VLGAVFGFSSYGPIALFGVIANESAP
PNLCGTSHAIVGLMANVGGFL-AG
aaaaaaaaaa aaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaa
410 420
430 440 450 hs1pw4a ( 402 )
aiVGytvdffgwdgGfmvMigGSilAvilLivVmigekrrheqllqelv
p QUERY g6pt LPFSTIAKHYSWSTAFWVAEVICAA
STAAFFLLRNIRTKMGRVSKKAE--
aaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa33333
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MPSA - only template and target
P_1P4W FKPAPHKARLPAAEIDPTYRRLRWQIFLG
IFFGYAAYYLVRKNFALAMPYLVEQG-FSRG GLUCOSE6HUMAN
-------------MAAQGYGYYRTVIFSAMFGGYSLYYFNRKTFSFV
MPSLVEEIPLDKD
. ..
.. P_1P4W DLGFALSGISIAYGFSKFIMGSV
SDRSNPRVFLPAGLILAAAVMLFMGFVPWATSSIAVM GLUCOSE6HUMA
N DLGFITSSQSAAYAISKFVSGVLSDQMSARWLFSSGLLLVG
LVNIFFAWS----STVPVF .
. .. ... .
. P_1P4W FVLLFLCGWFQGMGWPPCGRT
MVHWWSQKERGGIVSVWNCAHNVGGGIPPLLFLLGMAWF GLUCOSE6HU
MAN AALWFLNGLAQGLGWPPCGKVLRKWFEPSQFGTWWAILS
TSMNLAGGLGPILATI-LAQS .
. . . . .
P_1P4W NDWHAALYMPAFCAILVALFAFA
MMRDTPQSCGLP-----PIEEYKNDTAKQIFMQYVLP GLUCOSE6HUMA
N YSWRSTLALSGALCVVVSFLCLLLIHNEPADVGLRNLDPMP
SEGKKGSLKEESTLQELLL .
.. .. . ..
P_1P4W NKLLWYIAIANVFVYLLRYGILDW
SPTYLKEVKHFALDKSSWAYFLYEYAGIPGTLLCGW GLUCOSE6HUMAN
SPYLWVLSTGYLVVFGVKTCCTDWGQFFLIQEKGQSALVGSS
YMSALEVGGLVGSIAAGY .
. . . . .
. P_1P4W MSDKVFRGN--------RGATGVF
FMTLVTIATIVYWMNPAGN--PTVDMICMIVIGFLI GLUCOSE6HUMAN
LSDRAMAKAGLSNYGNPRHGLLLFMMAGMTVSMYLFRVTVTS
DSPKLWILVLGAVFGFSS .
. . .
P_1P4W YGPVMLIGLHALELAPKKAAGTAAGFT
GLFGYLGGSVAASAIVGYTVDFFGWDGGFMVMI GLUCOSE6HUMAN
YGPIALFGVIANESAPPNLCGTSHAIVGLMANVGGFLAGLPFSTI
AKHYSWSTAFWVAEV
. ... . . . . .
P_1P4W GGSILAVILLIVVMIGEKRRHEQLLQ
ELVP GLUCOSE6HUMAN ICAASTAAFFLLRNIRTKMGRVSK
KAE--- .
.
10
Extracted template-target
P_1PW4 -------FKPAPHKARLPAAEIDPTYRRLRWQIFLGI
FFGYAAYYLVRKNFALAMPYLVE gi2765461e
--------------------MAAQGYGYYRTVIFSAMFGGYSLYYFNRKT
FSFVMPSLVE
. P_1PW4
QGFS---RGDLGFALSGISIAYGFSKFIMGSVSDRSNPRVFLPAGLILAA
AVMLFMGFVP gi2765461e EIPLD--KDDLGFITSSQSAAYAISK
FVSGVLSDQMSARWLFSSGLLLVGLVNIFFAWSS
. . .
P_1PW4 WATSS--IAVMFVLLFLCGWFQGMGWPP
CGRTMVHWWSQKERGGIVSVWNCAHN--VGGG gi2765461e
TVP------VFAALWFLNGLAQGLGWPPCGKVLRKWFEPSQFGTWWAILS
TSMN--LAGG .
. .. P_1PW4
IPP-------LLFLLGMAWFN-----------DWHAALYMPAFCAILVAL
FAFAMMRDTP gi2765461e LGP-------ILATILAQSYS-----
-------WRSTLALSGALCVVVSFLCLLLIHNEP
. ..
P_1PW4 QSCGLPPIEEYKNDT-------------
------AKQIFMQYVLPNKLLWYIAIANVFVY gi2765461e
ADVGLRNLDPMPSEG--------------KKGSLKEESTLQELLLSPYLW
VLSTGYLVVF
. . . P_1PW4
LLRYGILDWSPTYLKEVKHFALDK-SSWAYFLYEYAGIPGTLLCGWMSDK
VFR------- gi2765461e GVKTCCTDWGQFFLIQEKGQSALV-G
SSYMSALEVGGLVGSIAAGYLSDRAMAKAGLSNY
. .
P_1PW4 -GNRGATGVFFMTLVTIATIVYWMNPAG
---------------NPTVDMICMIVIGFLIY gi2765461e
GNPRHGLLLFMMAGMTVSMYLFRVTVTSD-----------S--PKLWILV
LGAVFGFSSY
P_1PW4
GP-VMLIGLHALELAPKKAAGTAAGFTGLFGYLGGSVAASAIVGYTVDF-
FGWDGGFMVM gi2765461e GP-IALFGVIANESAPPNLCGTSHAI
VGLMANVG-GFLAGLPFSTIAKH-YSWSTAFWVA

P_1PW4 IGGSILAVILLIVVMIGEKRRHEQLLQE
LVP----------------------------- gi2765461e
EVICAASTAAFFLLRNIRTKMGRVSKKAE---------------------
----------
11
Checking alignment in MODELER

• Using chk_align.top script
• _aln.pos 210 220 230 240
  250 260 270
• 1PW4 MRDTPQSCGLPPIEEYKND/T-----AKQIFMQYVLPNKL
  LWYIAIANVFVYLLRYGILDWSPTYLKE
• G6PT IHNEPADVGLRNLDPMPSE-GKKGSLKEESTLQELLLSPY
  LWVLSTGYLVVFGVKTCCTDWGQFFLIQ
• _consrvd
  
• Problem near chain break
• _aln.pos 210 220 230 240
  250 260 270
• 1PW4 MRDTPQSCGLPPIEEYKND/----TAKQIFMQYVLPNKLL
  WYIAIANVFVYLLRYGILDWSPTYLKEV
• G6PT IHNEPADVGLRNLDPMPSEGKKGSLKEESTLQELLLSPYL
  WVLSTGYLVVFGVKTCCTDWGQFFLIQE
• _consrvd
  

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Modeler Runs

• Using extracted template and target alignment
• Sequence for template extracted from structure
  using Insight
• Missing residues in structure appear as chain
  breaks
• Parameters
• OUTPUT_CONTROL 1 1 1 1 1
• STARTING_MODEL 1
• ENDING_MODEL 5
• LIBRARY_SCHEDULE 4
• MD_LEVEL 'refine_1'

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PROSA 2 runs

• Used to evaluate models
• Models with best scores from MODELER were
  compared using PROSA
• Z value used for initial comparison
• Graph used to identify location of major
  violations

14
Model Selection Criteria

• MODELER log file
• Minimum energy
• Number of violations
• Number of really bad violations
• Location of violations with respect to alignment
  and structure
• PROSA 2 log file
• Z score closest to template
• Peaks and troughs in graph relative to template

15
Adjusting the alignment

• Comparison of structures obtained from modeler in
  Insight
• Alignment violations clearly visible
• Criteria for modifying alignment
• Unequal number of residues in loop
• Unsatisfied structural similarity constraints
• Residues violating constraints as generated by
  modeler

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1st run - adjustment in Insight
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Loop Modeling

• Modeler Run 2
• Loop Modeling Run 1

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Loop modeling

• Generate models based on adjusted alignment
• 25 models obtained
• Models selected based on minimum energy and
  constraint violations
• Parameters
• OUTPUT_CONTROL 1 1 1 1 1
• STARTING_MODEL 1
• ENDING_MODEL 5
• LIBRARY_SCHEDULE 2
• MD_LEVEL 'refine_3
• DO_LOOPS 1
• LOOP_ENDING_MODEL 5
• LOOP_MD_LEVEL 'refine_3

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Loop Modeling Run 1Best 4 Models Picked

• ID1, ID2
  1 5
• Current energy
  192
• PROSA Z score -6.60
• ( Z score of template -7.3 )
• ID1, ID2
  3 2
• Current energy
  387
• PROSA Z score -6.57
• ID1, ID2
  4 2
• Current energy
  363
• PROSA Z score -6.76
• ID1, ID2
  5 4
• Current energy
  242
• PROSA Z score -6.3

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Violations - MODELER log file
ID1, ID2
1 5 Current energy
192.1849
RESTRAINT_GROUP NUM
NUMVI NUMVP RMS_1 RMS_2 MOL.PDF
S_i ----------------------------------------------
--------------------------------------------------
- 25 Phi/Psi pair of dihedral restraints 64
44 11 36.170 140.638 79.036 1.000
--------------------------------------------------
----------------------------------------------- F
eature 25 Phi/Psi
pair of dihedral restraints List of the
RVIOL violations larger than 6.5000
ICSR RESNO1/2 ATM1/2 INDATM1/2 FEAT
restr viol rviol RESTR VIOL RVIOL
7 1360 45D 46K C N 368 370 -68.99
-70.20 30.80 2.20 -62.90 150.55 19.23
7 46K 46K N CA 370 371 109.62
140.40 -40.80 8 1361 46K
47D C N 377 379 173.18 54.50 123.21
12.43 -63.30 132.44 18.20 8 47D
47D N CA 379 380 7.79 40.90
-40.00 9 1362 47D 48D C N 385
387 -138.58 -63.30 76.02 11.52 -63.30
76.02 11.52 9 48D 48D N CA
387 388 -29.45 -40.00 -40.00
12 1369 103F 104A C N 811 813 -69.81
-68.20 21.24 1.77 -62.50 165.18 26.73
12 104A 104A N CA 813 814 124.12
145.30 -40.90 13 1370 104A
105A C N 816 818 -169.75 -62.50 107.58
21.02 -62.50 107.58 21.02 13 105A
105A N CA 818 819 -49.29 -40.90
-40.90
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1st loop model - violations in Insight
Residue 104
Residue 46
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Loop Model Run 1 - adjustment
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Loop Modeling 2

• Refinement of Loop Model 1
• Loop Modeling 2
• Modeler Run 3

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Loop Modeling Run 2Best 5 Models

• ID1, ID2
  5 1
• Current energy
  237.4322
• PROSA Z score -5.82
• ID1, ID2
  3 1
• Current energy
  222.2522
• PROSA Z score -6.27
• ID1, ID2
  1 1
• Current energy
  195.7286
• PROSA Z score -6.32
• ID1, ID2
  2 4
• Current energy
  226.8002
• PROSA Z score -6.09
• ID1, ID2
  2 2
• Current energy
  198.0359
• PROSA Z score -6.15

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Violations - MODELER log file
ID1, ID2
1 1 Current energy
195.7286
RESTRAINT_GROUP NUM
NUMVI NUMVP RMS_1 RMS_2 MOL.PDF
S_i ----------------------------------------------
--------------------------------------------------
- 4 Stereochemical improper torsion pot 156
1 2 1.943 1.943 16.723
1.000 25 Phi/Psi pair of dihedral restraints
67 40 11 34.260 132.074 73.358
1.000
--------------------------------------------------
----------------------------------------------- F
eature 25 Phi/Psi
pair of dihedral restraints List of the
RVIOL violations larger than 6.5000
ICSR RESNO1/2 ATM1/2 INDATM1/2 FEAT
restr viol rviol RESTR VIOL RVIOL
3 1430 45D 46K C N 368 370 -103.79
-118.00 33.92 1.76 -62.90 154.80 22.53
3 46K 46K N CA 370 371 169.89
139.10 -40.80 4 1431 46K
47D C N 377 379 -95.02 -70.90 59.16
2.00 -63.30 119.95 16.85 4 47D
47D N CA 379 380 -155.68 150.30
-40.00 5 1432 47D 48D C N 385
387 -63.33 -70.90 31.08 1.19 -63.30
160.16 19.77 5 48D 48D N CA
387 388 120.16 150.30 -40.00
9 1441 103F 104A C N 811 813 -122.41
-134.00 20.39 1.24 -62.50 166.47 30.50
9 104A 104A N CA 813 814 163.78
147.00 -40.90 10 1442 104A
105A C N 816 818 -64.90 -68.20 29.69
2.28 -62.50 156.71 25.57 10 105A
105A N CA 818 819 115.80 145.30
-40.90
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Loop Model Violation Sites
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Refinements in Final Model

• Some regions can be realigned and refined further
  taking into consideration their energy
  violations.
• Other tools could be used such as PROCHECK etc in
  addition to Modeler and PROSA to get further
  insight into energy details.
• Structural alignment of model with other known
  transport protein structures might be of some
  help.