Exploring the Biology of Disulfide-Rich
Hyperthermophiles through Protein
Phylogenetic Profiles
Navapoln Ramakul1, Morgan Beeby12, and Todd O. Yeates123
1Department
of Chemistry and Biochemistry, 2Department of Energy Center for Genomics and Proteomics, and
3Molecular Biology Institute, University of California, Los Angeles, CA 90095-1569
Southern California Bioinformatics Summer Institute
UCLA Bioinformatics:
Yeates Lab
• Goals: determine and analyze the threedimensional structures of proteins.
• Research: focus on protein structure & function,
protein sequence & evolution, and protein
assembly & design.
• Methods: crystal structure determination
through theoretical and computational methods.
General Overview
• Genomic Databases: create opportunities for
new kinds of computational analyses and novel
discoveries.
• Advantage: special comparative studies using
multiple genomes to compare sequence vs.
structure.
• Present Research: investigate the surprising
revelation about disulfide bonds in certain
microbes from comparative studies.
Protein Disulfide Bonds
• Intro
• Comput.
Methods
• Results &
Significance
• Applications
& Future
Directions
• Previously believed to be prominent only
outside the cell.
Inside the cell
• Disulfides only rarely found.
• Disulfides are transient or functionally
important, rather than stabilizing.
• Summary
Outside the cell
• Abundant.
Recent Studies
• Intro
• Comput.
Methods
• Results &
Significance
• Unexpected disulfides in an intracellular protein.
• Crystal structure of adenylosuccinate lyase
(ASL) from the P. aerophilum surprisingly
shown a protein chain stabilized by three
disulfide bonds (Toth et al., JMB (2000) 301,
433-450.).
• Applications
& Future
Directions
Disulfide bond
• Summary
Disulfide bond
Toth et al., JMB (2000) 301, 433-450
Evidence for Abundant S-S bonds
in P. aerophilum
• Intro
• Comput.
Methods
• Results &
Significance
Proteins with Even # of Cysteines
• Applications
& Future
Directions
• Summary
Mallick, Boutz, Eisenberg, and Yeates (2002). PNAS 99, 9679-9684
Disulfide Abundance in Various
Genomes
Genome
S)
Archaeal branch
f (S-
Pyrobaculum aerophilum
0.44
Aeropyrum pernix
0.40
Pyrococcus abyssi
0.31
Pyrococcus horikoshii
0.28
Aquifex aeolicus
0.17
Genome
f (S-S)
Pyro. aerophilum
0.44
104°C
Aero. pernix
0.40
100°C
Pyro. abyssi
0.31
102°C
Pyro. horikoshii
0.28
102°C
Aqui. aeolicus
0.17
93°C
Meth. thermo
0.15
90°C
Methanobacterium thermo 0.15
Thermotoga maritima
0.13
Methanococcus jannasc
0.13
Archaeoglobus fulgidus
0.11
Mycoplasma genitalium
0.06
Synechocystis PCC6803
0.08
Ureaplasma urealyticum
0.07
Neisseria meningitidis
0.06
Mycobacterium tubercu
0.07
Rickettsia prowazekii
0.06
Haemophilus influenzae
0.05
Escherichia coli
0.05
Treponema pallidum
0.03
Helicobacter pylori
0.03
Bacillus subtilis
0.01
90°C
86°C
92°C
Blue = archaea
= thermophile
Eubacterial branch
Mallick, Boutz, Eisenberg, and Yeates (2002). PNAS 99, 9679-9684
Exploring disulfide-rich
hyperthermophiles
• Intro
• Comput.
Methods
• Results &
Significance
• Applications
& Future
Directions
• Summary
• Find the sequences of glutaredoxin-like protein
in different organisms.
• Investigate the glutaredoxin-like protein in those
disulfide-rich hyperthermophiles.
• Goals: differences between glutaredoxin-like
protein in hyperthermophiles and glutaredoxin
in organisms.
Why glutaredoxin-like protein?
• Only present among hyperthermophiles.
• Operates in thiol-disulfide reaction via CXXC motif which either form
a disulfide (oxidized form) or a dithiol (reduced form).
• Requires for many functions including electron and proton transport
to essential enzymes like ribonucleotide reductase.
• Involves in formation of disulfide bonds in protein folding.
90o
Prototypical fold: E.coli thioredoxin (2TRX.pdb)
Methods
• Intro
• Comput.
Methods
• The sequences used in this study were obtained from
the National Center for Biotechnology Information
(www.ncbi.nlm.nih.gov).
• Results &
Significance
• Obtain the control sequence of glutaredoxin (E. coli) to
find glutaredoxin-like protein.
• Applications
& Future
Directions
• Search for the glutaredoxin-like protein sequences of
hyperthermophilic archaea.
• Summary
• Using Sequence-Structure Mapping to identify potential
disulfide bonds.
• Compare and analyze using multiple sequences
alignment program, such as ClustalW, T-Coffee, or
MSA.
Results
• ClustalW multiple sequences alignment of these glutaredoxin-like
proteins shows two CXXC motifs Green = P. aerophilum
Black = Hyperthermophilic archeae
Blue = Bacteria
= CXXC motif
Pyrobaculum_aerophilum
Aeropyrum_pernix
Pyrococcus_abyssi
Pyrococcus_horikoshii
Aquifex_aeolicus
Thermotoga_maritima
Glutaredoxin_1
Glutaredoxin_3
Bacillus_subtilis
Prim.cons.
10
20
30
40
50
60
70
80
90
100
110
120
130
140
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MAVPIGGPEEVPHIEVDEETKEIIKEMLSQMENPVNINFFTSPNCAGRETNWCIPTEELLDLLVQLAPQ-----GKLI-VNKYNAEKDVEAFKKFGVEPQRVPVIYFGE--GF-IRYLGAPMGEEVRAFIETVVRLSTGK
MAR-------YYVLDLSEDFRRELRETLAEMVNPVEVHVFLSK—SGCET--CEDTLRLMKLFEEESPTR--NGGKLLKLNVYYRESDSDKFSEFKVE--RVPTVAFLG--GE-VRWTGIPAGEEIRALVEVIMRLSEDE
----------MGLISEEDKRI-IKEEFFSKMVNPVKLIVFIG----KEHCQYCDQLKQLVQELSELT------DKLSYEIVDFDTPEGKELAEKYRIDRAP-ATTITQDGKDFGVRYFGIPAGHEFAAFLEDIVDVSRAE
----------MGLISEEDKRI-IKEEFFSKMVNPVKLIVFIG----KEHCQYCDQLKQLVQELSELT------DKLSYEIVDFDTPEGKELAEKYRIDRAP-ATTITQDGKDFGVRYFGIPAGHEFAAFLEDIVDVSKGD
------------MLLNLDVRMQLKELAQKEFKEPVSIKLFS----QAIGCESCQTAEELLKETVEVIGEAVGQDKIKLDIYSPFT--HKEETEKYGVDRVP-TIVIEGD-KDYGIRYIGLPAGLEFTTLINGIFHVSQRK
----------MGILSDKDIAY-LKDLFGKELKRKVKIVFFKTE--DKTRCQYCEITEQVLEELVSVD------PKLELEIHDFDS--DKEAVEKYQVEMVPATILLPEDGKDYGIRFYGVPSGHEFGTLIQDIITVSEGK
----------------------------------MQTVIFG-----RSGCPYCVRAKDLAEKLSNER--------------------DDFQYQYVDIRA---EGITKED----LQQKAGKPV-ETVPQIFVDQQHIGGYT
---------------------------------MANVEIYT-----KETCPYCHRAKALLS-----S--------------------KGVSFQELPIDG---NAAKREE----MIKRSGR---TTVPQIFIDAQHIGGCD
----------------------------------MRLIKLE-----QPNCNPCKMVSNYLEQVN------------------------IQ-FETVDVTQ---EPEVAAR-----FGVMGVP----VTILLSDQGEEVNRS
*
.
.
:
*
. :.
MA2PIGGPEEMGL2SEEDKRI3IKEEF2SEMVNPVK2IVF223NC4KE3CQYC222KQLLEEL2EL2P32VG2DKL2LEI2DFDT2EDKE2FEKY3VDR2PV4T3I2EDGKDFGIRYFGIPAG2E24AL2EDIVHVS2GK
Two CXXC motifs
Pyrobaculum_aerophilum
Aeropyrum_pernix
Pyrococcus_abyssi
Pyrococcus_horikoshii
Aquifex_aeolicus
Thermotoga_maritima
Glutaredoxin_1
Glutaredoxin_3
Bacillus_subtilis
Prim.cons.
150
160
170
180
190
200
210
220
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260
270
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TGLRQKTRAE-LSTLAQGAPKRVYILTVVTPSCPYCPYAVLMANMFAYES--KGK--VVSVVVEAYENPDIADMYGVTGVPTVILQAEDAAVGDVEFVGVPPEHELLA-------RVKNHMG--LS------------SGLEDATKEA-LKSLKG----RVHIETIITPSCPYCPYAVLLAHMFAYEAWKQGNPVILSEAVEAYENPDIADKYGVMSVPSIAIN------GYLVFVGVPYEEDFLD-------YVKSAAEGRLTVKGPIRAGEAEEL
TDLMAESKEE-VAKIDKN----VRILVFVTPTCPYCPLAVRMAHKFAIENTKAGKGKILGDMVEAIEYPEWADQYNVMAVPKIVIQVDGE--DKVQFEGAYPEKMFLE-------KLLAALS----------------TDLMQDSKEE-VSKIDKD----VRILIFVTPTCPYCPLAVRMAHKFAIENTKAGKGKILGDMVEAIEYPEWADQYNVMAVPKIVIQVNGE--DKVQFEGAYPEKMFLE-------KLLSALS----------------PQLSEKTLEL-LQVVDIP----IEIWVFVTTSCGYCPSAAVMAWDFALAN-----DYITSKVIDASENQDLAEQFQVVGVPKIVINKG-----VAEFVGAQPENAFLGYIMAVYEKLKREKEQA--------------PQLSEESIQK-LQSLEEP----IRISVFVTPTCPYCPRAVLMAHNMAMAS-----DKIIGEMIEANEYWELSEKFGVSSVPHIVVNRDP----SKFFVGAYPEKEFIN------EVLRLAKG----------------DFAAWVKEN--LDA----------------------------------------------------------------------------------------------------------------------------DLYALDARGG-LDPLLK-------------------------------------------------------------------------------------------------------------------------VGFKPNELDELLKELR--------------------------------------------------------------------------------------------------------------------------:
TGL3232KEELL42LDKPAPKRVRILVFVTP2CPYCP2AVLMAH2FA2ENTKAGK2KIL22MVEA2E2P23ADQYGVM3VPKIVI2VDGEAV2KV2FVGAYPEK2FLEYIMAVYEKLKSA2322L2VKGPIRAGEAEEL
• Glutaredoxin-like protein has more than 85 amino acids.
Results
• Intro
• Comput.
Methods
• Results &
Significance
• Applications
& Future
Directions
• Most organisms have 1 CXXC
motif in glutaredoxin.
• Glutaredoxin-like protein has two
redox-active CXXC motifs per
polypeptide.
• Exception: P. aerophilum has only
1 CXXC motif.
P. furiosus:
P. horikoshii:
• Summary
CXXC motifs
1A8L.pdb, Nat. Struct. Biol. (1998), 5 (7) 602-611
1J08.pdb, unpublished
Limitation
• Intro
• Comput.
Methods
• Results &
Significance
• Applications
& Future
Directions
• Summary
• Only 25 genomes.
• Some glutaredoxin-like proteins
have not yet been sequenced.
Applications and Further Studies:
• Intro
• Comput.
Methods
• Results &
Significance
• Applications
& Future
Directions
• Summary
• How disulfide bonds involve in protein folding?
• To identify disulfide-bonded protein-protein
interactions and networks.
• To investigate the stability mechanisms by
disulfide bonds.
Summary
• Intro
• Comput.
Methods
• Results &
Significance
• Applications
& Future
Directions
• Summary
• Most of the hyperthermophiles have 2 CXXC motifs in
order to have abundant disulfide bonds.
• The abundance of disulfide bonds appear to play a key
role in stabilizing protein at high temperature.
• Intracellular disulfide bond is a characteristic of all
archaea or an adaptation to high temperature.
• This study illustrates the power of integrating genomic
data with protein structure and function to illuminate
the chemistry and biology of unusual organisms.
Acknowledgements
Yeates Lab
Dr. Todd Yeates
Morgan Beeby
Everyone else at Yeates lab
CalState LA
Research mentors
SoCalBSI Program
SoCalBSI interns
Support
National Science Foundation (NSF)
National Institutes of Health (NIH).
UCLA-DOE Center for Genomics and Proteomics