proteases jessica bell advanced biochemistry class university of richmond march 12th, 2002
TRANSCRIPT
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Proteases
Jessica BellAdvanced Biochemistry Class
University of RichmondMarch 12th, 2002
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AdhesionP. gingivalis protease
Immune Response
T-cell protease
Reproduction and
Fertilizationacronase
Tumor Invasion
collagenase
Coagulationthrombin
Digestiontrypsin
Blood pressure regulation
renin
Secretionsignal peptidases
Developmentsnake
Complement Fixation
CI protease
Fibrinolysistissue
plasminogen actvator
Hormone Processing
Kex 2
Animal Virus ReplicationHIV protease
Pain Sensingkallikrein
Cell fusionhemaglutinase
6 Broad Categories
Function Protease
Nutrition trypsin subtilisin, a-lytic protease
Invasion matrix metallo proteases
Evasion IgA protease
Adhesion P. gingivalis protease
Processing signal peptidase, viral proteases, proteosome
Signaling caspases, granzymes
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Biology Differs, Chemistry is the Same
endopeptidase
exopeptidase
+3HN C
O
C COO-N C
R1 R2H
H H
+ H2O
+3HN C COO-
R1
H
+3HN C COO-
R2
H+
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Michaelis Complex
O
H
195
N N:
H
57
O
O
102
O
HN
P1
-
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Michaelis Complex
Transition State to Acylation
O
H
195
N N:
H
57
O
O
102
O
HN
P1
-
O
H
195
N NH
57
O
O
102
O
HN
P1
+-
-
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Michaelis Complex
Transition State to Acylation
Tetrahedral Intermediate I
O
H
195
N N:
H
57
O
O
102
O
HN
P1
-
O
H
195
N NH
57
O
O
102
O
HN
P1
+-
-
OH
195
N NH
57
O
O
102
O -
HN
P1
- +
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Michaelis Complex
Transition State to Acylation
Tetrahedral Intermediate I
O
H
195
N N:
H
57
O
O
102
O
HN
P1
-
O
H
195
N NH
57
O
O
102
O
HN
P1
+-
-
OH
195
N NH
57
O
O
102
O -
HN
P1
- +
Acyl-Enzyme
O
195
N N:
H
57
O
O
102
O
NH3+
P1
-
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Michaelis Complex
Transition State to Acylation
Tetrahedral Intermediate I
O
H
195
N N:
H
57
O
O
102
O
HN
P1
-
O
H
195
N NH
57
O
O
102
O
HN
P1
+-
-
OH
195
N NH
57
O
O
102
O -
HN
P1
- +
Acyl-Enzyme
O
195
N N:
H
57
O
O
102
O
NH3+
P1
-
Transition State to Deacylation
O
195
N NH
57
O
O
102
O
P1
H
OH
- +-
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Michaelis Complex
Transition State to Acylation
Tetrahedral Intermediate I Acyl-Enzyme
Transition State to Deacylation
Tetrahedral Intermediate II
O
H
195
N N:
H
57
O
O
102
O
HN
P1
-
O
H
195
N NH
57
O
O
102
O
HN
P1
+-
-
OH
195
N NH
57
O
O
102
O -
HN
P1
- +O
195
N N:
H
57
O
O
102
O
NH3+
P1
-
O
195
N NH
57
O
O
102
O
P1
H
OH
- +-
O
195
N NH
57
O
O
102
O -
P1
H
OH
- +
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Mechanistic Sets of Proteasesset feature inhibitor examples function
serine protease active site serine fluorophosphates trypsin digestionH57, D102, S195 thrombin blood coagulation
plasmin lysis of blood clotscoccoonase mechanicalsubtilisin digestionacrosin sperm penetration
metalloproteases Zn2+, E270 o-phenanthroline carboxypeptidase digestionZn2+, Ca2+ o-phenanthroline thermolysin digestionE143, H231
sulfhydryl protease active site cysteine iodoacetate papain digestionC25, H159, N175 strept. proteinase digestion
cathepsin B intracell. digestion
Acid protease acidic pH optimum diazoketones pepsin digestionD32, D215 chymosin milk coagulation
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Trypsin Elastase Subtilisin
Same Fold
Catalytic Triad Conserved
Divergent vs. Convergent Evolution
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Carboxypeptidase A
Thermolysin
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Pepsin
HIV Protease
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NH
HN
NH
HN
NH
OH
NH3+
CH3
OH
OO
O O
Peptide
Subsite of Protease
P2 P1 P1’ P2’
S2 S1 S1’ S2’
ScissileBond
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Substrate Selection within the Same Tertiary Fold
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Ecotin
Serine Protease Inhibitor
Unknown function
Dimeric
1° and 2° binding sites
Cleaved
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Cystatin Superfamily
Cysteine protease inhibitors
Non-canonical binding
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SerpinsSerine protease
inhibitors
Irreversible
Disruption of 3º structure
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CH2 C
CH3
H O
C N C
H
C
O
O
H2N
H2N
C NH
H
AZn2+
S
Arg
NH C
O
C N C
R2 R1H
H H O
C N C
R1H
H
C
O
O
H2N
H2N
C NH
H
AZn2+
Arg- +
+-
Captopril
carboxy-di-peptidase active site
H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-His-Leu-Co2H
H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-Co2H
Proangiotensin
Angiotensin
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Ways to Determine Specificity
Synthesis of short peptides [15 to 20a.a.], check for cleavage with PAGE
Phage display of short peptides
Positional scanning synthetic combinatorial libraries [PS-SCL]
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NH
HN
NH
HN
OO
O OX
X
X
X
HN
O
Ac-XXXO-AMC A R N D E Q G H I LK F P S T W Y V mA R N D E Q G H I LK F P S T W Y V mA R N D E Q G H I LK F P S T W Y V mA R N D E Q G H I LK F P S T W Y V m
Ac-XXOX-AMC
Ac-OXXX-AMC
Ac-XOXX-AMC
7-amino-4-methyl coumarin
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0.044
0.046
0.048
0.05
0.052
0.054
0.056
0.058
0.06
A R N D Q E G H I L K M m F P S T W Y V
0.0
100.0
200.0
300.0
400.0
500.0
A R N D Q E G H I L K F P S T W Y V m M0.0
50.0
100.0
150.0
200.0
A R N D Q E G H I L K F P S T W Y V m M
0.0
100.0
200.0
300.0
400.0
A R N D Q E G H I L K F P S T W Y V m M
NH
HN
NH
HN
OO
O OP4
P3
P2
P1
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Cytotoxic Lymphocytes
Molecular Biology of the Cell, Garland
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Cytotoxic T Lymphocyte Apoptotic Pathway
3 Fas
DDFADD
DED
aggregrates pro-caspase 8, intermolecular cleavage to caspase 8, activation of effector caspases [3, 6, 7],
apoptosis
MPR?
Granzymes
Perforin
nucleus
cleave pro-caspases
apoptosis
GrnB GrnA
Nuclease?
Single stranded breaks in DNA
Mito.
Bcl-2
Cytotoxic T lymphocyte
serpins
Ca2+
Ca2+
Ca2+
Ca2+
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Granzymes: Lymphocyte Serine ProteasesName Activity Predicted P1 MW
cleavage site
A Trypsin-like R/K 60 (Dimer)
B Asp-ase D/E 35
C Unknown N/S 27
D Unknown F/L 35-50
E Unknown F/L 35-45
F Unknown F/L 35-40
G Unknown F/L
H Chymase F
I Unknown
J Unknown
K Trypsin-like 30
M Met-ase M/L/nor-L 30
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Granzyme Structure
Waugh et al. (2000) Nat. Struct. Biol. 7:762-765
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Granzyme A, Proposed Dimeric Structure
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Granzyme A: Substrate Specificity and Macromolecule Substrates
Substrate Sequence P4 P3 P2 P1
FLUOROGENIC LIBRARIES V/I G/A/S N R
PIL-1 D A P V R S L N C T
THROMBIN RECEPTOR T L D P R S F L L R
HISTONE H1 K L G L K S L V S K
HISTONE H2b A P A P K K G S K K
SET Q T Q N K A S R K R
LAMIN B V T V S R A S S S R
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Research AimsQuaternary Structure and Substrate Selectivity
Crystallographic Study
Dimer vs. Monomer Cell Lysate Assays
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0
0.2
0.4
0.6
0.8
1
1.2
mO
D/m
in @
405
nm
[Inhibitor], M
0 0.05 5 50
Macromolecular Inhibition of Granzyme A
Control
mM84R Eco
dM84R Eco
Tryp. Inh.
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Potential Effects of Oligomer on Macromolecular Inhibitors
grnA
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Potential Effects of Oligomer on Macromolecular Inhibitors
grnB:dEcotin
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Potential Effects of Oligomer on Macromolecular Inhibitors
mEcotin
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Small Molecule Inhibitor of Granzyme Am
OD
/min
ute
@40
5nm
[Inhibitor], nM
0 50 100 150 200
N C
O
C C N C C
O
O
N C CH2Cl
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Box1 WellB3 Box1 WellC3 Box1 WellC5
Box1 WellD2 Box2 WellC4
Box3 WellC5
Box4 WellA2Box2 WellC4a
Crystallization Trials I
Citrate pH5.6/Acetate/PEG4K Cac. pH6.5/AmSO4/PEG8K Tris pH8.5/LiSO4/PEG4K Citrate pH6.5/t-Butanol
MES pH6.5/AmSO4/PEG-me5KCitrate pH6.5/i-propanol/PEG4KAcetate pH4.5/AmSO4/PEG4K
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Citrate pH5.6/Acetate/PEG4K Citrate pH6.5/i-prop/PEG4K
Crystallization Trials II
30% 20%
10%20%
25% 15%
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Diffraction!!!
Unit Cell a=72.85 b=175.9 c=152.55, Å
=99.24º
<I/I> 14.3
redundancy 90% of the data is measured ≥2X
Resolution 25-2.55Å
completeness 97.5% [89%]
[2.64-2.55Å shell]
chi2 1.2
Rmerge 0.049
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Research AimsQuaternary Structure and Substrate Selectivity
Crystallographic Study
Dimer vs. Monomer Cell Lysate Assays
Substrate Selection amongst Isozymes
Structural Comparison of Human and Mouse
Mutagenesis & Substrate Libraries
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Granzyme A: Human and MouseHuman MRNSYRFLAS SLSVVVSLLL IPEDVCEKII GGNEVTPHSR PYMVLLSLDRMouse MRNASGPRGP SLATLLFLLL IPEGGCERII GGDTVVPHSR PYMALLKLSS
Human KTICAGALIA KDWVLTAAHC NLNKRSQVIL GAHSITREEP TKQIMLVKKEMouse NTICAGALIE KNWVLTAAHC NVGKRSKFIL GAHSINK-EP EQQILTVKKA # Human FPYPCYDPAT REGDLKLLQL TEKAKINKYV TILHLPKKGD DVKPGTMCQVMouse FPYPCYDETT REGDLQLVRL KKKATVNRNV AILHLPKKGD DVKPGTRCRV #Human AGWGRTHNSA SWSDTLREVN ITIIDRKVCN DRNHYNFNPV IGMNMVCAGSMouse AGWGRFGNKS APSETLREVN ITVIDRKICN DEKHYNFHPV IGLNMICAGD
Human LRGGRDSCNG DSGSPLLCEG VFRGVTSFGL ENKCGDPRGP GVYILLSKKHMouse LRGGKDSCNG DSGSPLLCDG ILRGITSFG- GEKCGDRRWP GVYTFLSDKH # * *Human LNWIIMTIKG AVMouse LNWIKKIMKG SV 68% Identical!
P4 P3 P2 P1
Human V/I G/A/S N R
Mouse G F/Y F R
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Substrate Specificity of Granzyme A Species
D102H57
S195P1P2
P3P4
D189
R99
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Substrate Specificity of Granzyme A Species
P4
W224
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Substrate Specificity of Granzyme A Species
P4
W224
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Substrate Specificity of Granzyme A Species
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C. S. CraikCraik Lab Members
Granzyme ASandy Waugh Sami MahrusCarly Klein
MT-SP1Jeonghoon Sun
Ami Bhatt
R. J. FletterickFletterick Lab Members
Mary Jane Budny
and all those helpful people who...
ALS 8.3.1James Holton
NIH: The $$$ people