Adhesion P. gingivalis protease Immune Response T-cell protease Reproduction and Fertilization...
-
Upload
adele-gregory -
Category
Documents
-
view
232 -
download
1
Transcript of Adhesion P. gingivalis protease Immune Response T-cell protease Reproduction and Fertilization...
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, -lytic protease
Invasion matrix metallo proteases
Evasion IgA protease
Adhesion P. gingivalis protease
Processing signal peptidase, viral proteases, proteosome
Signaling caspases, granzymes
What do proteases do?
+3HN C
O
C COO-N C
R1 R2H
H H
+ H2O
+3HN C COO-
R1
H
+3HN C COO-
R2
H+
Uncatalyzed rxn at neutral pH, 37°C: 1 X 10-10 /sec
Catalyzed rxn (chymotrypsin) at neutral pH, 37°C: 100/sec
Conditions for chemically catalyzed reaction:
24hrs. @ 6M HCl, 110°C
Go for the rxn is -2kcal/mol
But…
Koshland, D. (1996) J. Cell. Comp. Phys. Suppl. 1 43:217.
endopeptidase
exopeptidase
Two types of cleavages
Same rxn, Four mechanisms
Named for residue/group in active site of enzyme essential for most effective catalysis
Serine -OH
Cysteine/Thiol -SH
Acid/Aspartic COO-
Metallo Zn2+
Mechanistic Sets of Proteases
set 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
Cysteine 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
Metalloproteases Zn2+, E270 o-phenanthroline carboxypeptidase digestionZn2+, Ca2+ o-phenanthroline thermolysin digestionE143, H231
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Serine Protease Mechanism – The players
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Serine Protease Mechanism – Oxyanion Hole
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.
Serine inhibitors
CH2
CH C
O
NHS
O
O
CH2 Cl
CH3
Peptide bond mimic
Chloro-methyl ketone [CMK]
TPCK
(L-1-Chloro-3-[4-tosylamido]-4-phenyl-2-butanone)
Serine inhibitors
DFP
Diisopropyl fluorophosphate
CH3
P O
O
CH
CH3
CH O
CH3
CH3
F
Trypsin Elastase Subtilisin
Same Fold
Catalytic Triad Conserved
Divergent vs. Convergent Evolution
SerpinsSerine protease
inhibitors
Irreversible
Disruption of 3º structure
Ecotin
Serine Protease Inhibitor
Unknown function
Dimeric
1° and 2° binding sites
Cleaved
Cysteine protease mechanism
S
H
25
N N:
H
159
O
HN
P1
SH
25
N NH
159
O -
HN
P1
+
S
25
N N:
H
159
O
NH2
P1
S
25
N NH
159
O -
P1
H
OH
+
Acyl Intermediate
Tetrahedral intermediate I
Tetrahedral intermediate II
Michaelis Complex
H2O
NH2
Cysteine protease mechanism
S
H
25
N N:
H
159
O
HN
P1
SH
25
N NH
159
O -
HN
P1
+
S
25
N N:
H
159
O
NH2
P1
S
25
N NH
159
O -
P1
H
OH
+
Acyl Intermediate
Tetrahedral intermediate I
Tetrahedral intermediate II
Michaelis Complex
H2O
NH2
Covalent Intermediate
No Asp102 equivalent
Cysteine protease inhibitors
CH2 C
O
OH
I
S
H
25
N N:
H
159
Iodoacetic acid
E-64
(2S,3S)-3-(N-(1S)-1-[N-(4guanidinobutyl)carbamoyl]3methylbutyl)carbamoyl)
oxirane-2-carboxylic acid
Cystatin Superfamily
Cysteine protease inhibitors
Non-canonical binding
Acid protease mechanism
OO
H
OH
O
NP1
H
P1’
H OO
H
O
H
-
O
O
O
NP1
H
P1’
H
OO
H O
H-
O
O
O-NP1
H
P1’
H
O-O
H-
O
O
N
H
P1’
H
O
P1
O
H
-
O
-O
Asp25 Asp25
Asp25 Asp25
Asp25’ Asp25’
Asp25’ Asp25’
Michaelis complex
Tetrahedral intermediate
Acid protease mechanism
OO
H
OH
-
O
-O
O
NP1
H
P1’
H OO
H
O
H
-
O
O
O
NP1
H
P1’
H
OO
H O
H-
O
O
O-NP1
H
P1’
H
O-O
H-
O
O
N
H
P1’
H
O
P1
O
H
Asp25 Asp25
Asp25 Asp25
Asp25’ Asp25’
Asp25’ Asp25’
Michaelis complex
Tetrahedral intermediate
No covalent intermediate
Activated water
Acid protease inhibitors
Indinavir, Roche
Reiling, K. K. et al. Biochemistry (2002) 41:4582-94.
RHN
HN
NNHR’
CH3
OH
OO
O
HIV Protease Substrate
Movie of Multi-drug resistant HIV Models:
www.ucsf.edu
Click on A-Z listings
Under C find Craik, Charles
Within the Craik website there is section entitled movies
Enjoy!
Pepsin
HIV Protease
Metallo protease mechanism
Zn2+His
Glu
HisH
O
H
Zn2+His
Glu
His
O
H
Zn2+
HisGluHis
O
-
O
-O
NH
P1’
H
O
P1
Zn2+
HisGluHis -
O
-O
O-
O
P1
Zn2+
HisGluHis
ON
P1
H
P1’
-
O
O
O
H
Zn2+
HisGluHis
O
-
O
OO
N
P1H
P1’
H
Metallo protease mechanism
Zn2+His
Glu
HisH
O
H
Zn2+His
Glu
His
O
H
Zn2+
HisGluHis
O
-
O
-O
NH
P1’
H
O
P1
Zn2+
HisGluHis -
O
-O
O-
O
P1
Zn2+
HisGluHis
ON
P1
H
P1’
-
O
O
O
H
Zn2+
HisGluHis
O
-
O
OO
N
P1H
P1’
H
No covalent intermediate
Activated water
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
Carboxypeptidase A
Thermolysin
Synopsis of Protease MechanismsSerine
Ser-His Asp Catalytic Triad
covalent intermediate
Cysteine
Cys-His
covalent intermediate
Acid
Asp-Asp
Activated water
no covalent intermediate
Metallo
Zn2+ or equivalent-Glu
Activated Water
no covalent intermediate
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
How Proteases Order Off the Menu
Substrate Selection within One Tertiary Fold
Methods to Determine Specificity
1> Synthesis of short peptides [15 to 20a.a.], check for cleavage with PAGE
2> Phage display of short peptides
3> Positional scanning synthetic combinatorial libraries [PS-SCL]
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
Harris J. L. et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic
substrate libraries. PNAS (2000) 97:7754-9.
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
Regulation of Proteases – A Few Examples
Zymogens
Pro-peptide that must be cleaved before protease becomes fully active
Enteropeptidase
Trypsinogen1 16
Trypsin16
1 15
Zymogen form has distorted oxyanion hole and substrate binding pocket
Compartmentalization
Macromolecular Inhibitors
Host and non-host
Cytotoxic Lymphocytes
Molecular Biology of the Cell, Garland
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+
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
Granzyme Structure
Waugh et al. (2000) Nat. Struct. Biol. 7:762-765
Granzyme A, Proposed Dimeric Structure
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
Chasing the Crystals
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.
Potential Effects of Oligomer on Macromolecular Inhibitors
grnA
Potential Effects of Oligomer on Macromolecular Inhibitors
grnB:dEcotin
Potential Effects of Oligomer on Macromolecular Inhibitors
mEcotin
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
CrystallizationPrevious conditions:
0.1M Citrate, pH 5.6, 20% peg 4K, 20% Isopropanol
New Conditions:
4M NaFormate
0.1M Citrate, pH 5.6, 20-30% peg4K, 0.2M AmAcetate
0.1M Cacodylate, pH 6.5, 15-20% peg4K, 0.2M AmSO4
0.1M Tris, pH8.5, 13-18% peg4K, 0.2M LiSO4
Diffraction!!!
Substrate Selectivity
Granzyme A: Human and Mouse
68% Identical!
P4 P3 P2 P1
Human V/I G/A/S N R
Mouse G F/Y F R
Human 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
Substrate Specificity of Granzyme A Species
D102H57
S195P1P2
P3P4
D189
R99
Substrate Specificity of Granzyme A Species
P4
W224
Substrate Specificity of Granzyme A Species
P4
W224
Substrate Specificity of Granzyme A Species
P1-Arg PS-SCL of Native Human GrA - P4
0
0.01
0.02
0.03
0.04
0.05
0.06
A R N D Q E G H I L K F P S T W Y V n
Amino Acid
Rel
ativ
e F
luor
esce
nce
Uni
ts
P1-Arg PS-SCL of Human GrA - P3
0
0.01
0.02
0.03
0.04
0.05
0.06
A R N D Q E G H I L K F P S T W Y V n
Amino Acid
Rel
ativ
e F
luor
esce
nce
Uni
ts
P1-Arg PS-SCL of Native Human GrA - P2
0
0.01
0.02
0.03
0.04
0.05
0.06
A R N D Q E G H I L K F P S T W Y V n
Amino Acid
Rel
ativ
e F
luor
esce
nce
Uni
ts
Native Human GrAHuman Mouse
P2 N F
P3 G/A/S F/Y
P4 V/L G
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
A R N D Q E G H I L K F P S T W Y V n
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
A R N D Q E G H I L K F P S T W Y V n
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
A R N D Q E G H I L K F P S T W Y V n
H -> M GrAHuman Mouse
P2 N F
P3 G/A/S F/Y
P4 V/L G
Conclusions: Mutational Studies
The residues identified from the model of mouse granzyme A [L201, G202, E203, W211] when mutated into the equivalent positions of the human homologue:
1> switch the substrate specificity at the P3 position,
2> increase the preference for small residues [A/G] over branched residues [I/V] at the P4 position and
3> broaden residue selection at the P2 position.
C. S. CraikCraik Lab Members
Granzyme ASandy Waugh Sami MahrusCarly Klein
MT-SP1Jeonghoon Sun
Ami Bhatt
The ChemistsAmy BarriosAlan Marnett
R. J. FletterickFletterick Lab Members
ALS 8.3.1James Holton
NIH: The $$$ people