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Proteases Jessica Bell Advanced Biochemistry Class University of Richmond March 12th, 2002. Adhesion P. gingivalis protease. Secretion signal peptidases. Immune Response T-cell protease. Development snake. Blood pressure regulation renin. Digestion trypsin. Coagulation thrombin.

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Proteases

Jessica Bell

Advanced Biochemistry Class

University of Richmond

March 12th, 2002


Slide2 l.jpg

Adhesion

P. gingivalis protease

Secretion

signal peptidases

Immune Response

T-cell protease

Development

snake

Blood pressure regulation

renin

Digestion

trypsin

Coagulation

thrombin

Complement Fixation

CI protease

Cell fusion

hemaglutinase

Tumor Invasion

collagenase

Reproduction and Fertilization

acronase

Pain Sensing

kallikrein

Fibrinolysis

tissue plasminogen actvator

Animal Virus Replication

HIV protease

Hormone Processing

Kex 2

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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H

O

H

+

H2O

+

+3HN

C

C

N

C

COO-

R1

H

R2

H

+3HN

C

COO-

R1

H

+3HN

C

COO-

R2

Biology Differs, Chemistry is the Same

endopeptidase

exopeptidase


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Michaelis Complex

57

195

-

O

N:

N

102

O

H

H

O

O

HN

P1


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Michaelis Complex

57

195

-

O

N:

N

102

102

O

O

H

H

O

O

O

HN

P1

57

195

O

d+

-

N

N

H

H

d-

O

HN

P1

Transition State to Acylation


Slide6 l.jpg

Michaelis Complex

57

195

-

O

N:

N

102

102

102

O

O

O

H

H

O

O

O

O

HN

P1

57

195

O

d+

-

N

N

H

H

d-

O

HN

P1

57

195

+

-

N

H

N

O

H

O-

HN

P1

Transition State to Acylation

Tetrahedral Intermediate I


Slide7 l.jpg

Michaelis Complex

57

195

-

O

N:

N

102

102

102

102

O

O

O

O

H

H

O

O

O

O

O

HN

P1

57

195

O

d+

-

N

N

H

H

d-

O

HN

P1

57

57

195

195

+

-

-

N:

N

H

N

N

O

O

H

H

O

O-

HN

NH3+

P1

P1

Transition State to Acylation

Acyl-Enzyme

Tetrahedral Intermediate I


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Michaelis Complex

57

195

-

O

N:

N

102

102

102

102

102

O

O

O

O

O

H

H

O

O

O

O

O

O

HN

P1

57

57

195

195

O

d+

d+

-

-

N

N

N

N

H

O

d-

H

H

H

d-

O

O

O

HN

H

P1

P1

57

57

195

195

+

-

-

N:

N

H

N

N

O

O

H

H

O

O-

HN

NH3+

P1

P1

Transition State to Deacylation

Transition State to Acylation

Acyl-Enzyme

Tetrahedral Intermediate I


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Michaelis Complex

Tetrahedral Intermediate II

57

57

195

195

+

-

-

H

N

N

O

O

N:

N

102

102

102

102

102

102

H

O-

O

O

O

O

O

O

H

H

O

O

O

O

O

O

O

O

H

HN

P1

P1

57

57

195

195

O

d+

d+

-

-

N

N

N

N

H

O

d-

H

H

H

d-

O

O

O

HN

H

P1

P1

57

57

195

195

+

-

-

N:

N

H

N

N

O

O

H

H

O

O-

HN

NH3+

P1

P1

Transition State to Acylation

Transition State to Deacylation

Tetrahedral Intermediate I

Acyl-Enzyme


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Mechanistic Sets of Proteases

set feature inhibitor examples function

serine protease active site serine fluorophosphates trypsin digestion

H57, D102, S195 thrombin blood coagulation

plasmin lysis of blood clots

coccoonase mechanical

subtilisin digestion

acrosin sperm penetration

metalloproteases Zn2+, E270 o-phenanthroline carboxypeptidase digestion

Zn2+, Ca2+ o-phenanthroline thermolysin digestion

E143, H231

sulfhydryl protease active site cysteine iodoacetate papain digestion

C25, H159, N175 strept. proteinase digestion

cathepsin B intracell. digestion

Acid protease acidic pH optimum diazoketones pepsin digestion

D32, D215 chymosin milk coagulation


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Divergent vs. Convergent Evolution

Catalytic Triad Conserved

Trypsin

Elastase

Subtilisin

Same Fold


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Thermolysin

Carboxypeptidase A


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Pepsin

HIV Protease


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OH

CH3

O

O

HN

HN

NH

NH

NH

O

O

OH

NH3+

P2

P1

P1’

P2’

Peptide

Scissile

Bond

Subsite of Protease

S2

S1

S1’

S2’



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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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Serpins

Serine protease inhibitors

Irreversible

Disruption of 3º structure


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A

Zn2+

H

H

O

H

O

H

O

H2N

NH

C

C

N

C

C

N

C

C

C

NH

Arg

R2

H

R1

H

R1

O

H2N

A

Zn2+

H

H

O

H

O

H2N

S

CH2

C

C

N

C

C

C

NH

Arg

CH3

O

H2N

H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-His-Leu-Co2H

Proangiotensin

H2N-Asp-Arg-Val-Tyr-Ile-Pro-Phe-Co2H

Angiotensin

-

+

carboxy-di-peptidase active site

+

-

Captopril


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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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X

O

O

X

HN

HN

NH

NH

X

X

O

O

HN

O

7-amino-4-methyl coumarin

R

N

D

E

Q

G

H

I

L

A

Ac-XXXO-AMC

K

F

P

S

T

W

Y

V

m

R

N

D

E

Q

G

H

I

L

A

Ac-XXOX-AMC

K

F

P

S

T

W

Y

V

m

R

N

D

E

Q

G

H

I

L

A

Ac-XOXX-AMC

K

F

P

S

T

W

Y

V

m

R

N

D

E

Q

G

H

I

L

A

Ac-OXXX-AMC

K

F

P

S

T

W

Y

V

m


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P2

O

O

P4

HN

HN

NH

NH

P1

P3

O

O


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Cytotoxic Lymphocytes

Molecular Biology of the Cell, Garland


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Cytotoxic T Lymphocyte Apoptotic Pathway

Cytotoxic T lymphocyte

Granzymes

Perforin

Ca2+

Ca2+

Ca2+

3

Fas

Ca2+

GrnA

GrnB

MPR?

DD

FADD

cleave

pro-caspases

serpins

Nuclease?

DED

Mito.

apoptosis

Single stranded breaks in DNA

Bcl-2

aggregrates pro-caspase 8, intermolecular cleavage to caspase 8, activation of effector caspases [3, 6, 7], apoptosis

nucleus


Slide25 l.jpg

Granzymes: Lymphocyte Serine Proteases

Name Activity Predicted P1 MW

cleavage site

A Trypsin-like R/K 60 (Dimer)

BAsp-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



Slide28 l.jpg

Substrate Sequence

P4 P3 P2 P1

FLUOROGENIC LIBRARIES V/I G/A/S N R

PIL-1b 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

Granzyme A: Substrate Specificity and Macromolecule Substrates


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Research Aims

Quaternary Structure and Substrate Selectivity

Crystallographic Study

Dimer vs. Monomer Cell Lysate Assays


Slide30 l.jpg

mOD/min @ 405nm

0

0.05

5

50

[Inhibitor], mM

Macromolecular Inhibition of Granzyme A

Control

mM84R Eco

dM84R Eco

Tryp. Inh.





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O

O

N

C

C

N

C

C

N

C

C

CH2Cl

O

Small Molecule Inhibitor of Granzyme A

mOD/minute @405nm

0

50

100

150

200

[Inhibitor], nM


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Box1 WellB3

Box1 WellC3

Box1 WellC5

Box3 WellC5

Box4 WellA2

Box1 WellD2

Box2 WellC4

Box2 WellC4a

Crystallization Trials I

Citrate pH5.6/Acetate/PEG4K

Cac. pH6.5/AmSO4/PEG8K

Tris pH8.5/LiSO4/PEG4K

Citrate pH6.5/t-Butanol

Acetate pH4.5/AmSO4/PEG4K

Citrate pH6.5/i-propanol/PEG4K

MES pH6.5/AmSO4/PEG-me5K


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Crystallization Trials II

Citrate pH5.6/Acetate/PEG4K

Citrate pH6.5/i-prop/PEG4K

30%

20%

20%

10%

25%

15%


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Diffraction!!!

Unit Cell a=72.85 b=175.9 c=152.55, Å

b=99.24º

<I/sI> 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 Aims

Quaternary 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 Mouse

Human MRNSYRFLAS SLSVVVSLLL IPEDVCEKII GGNEVTPHSR PYMVLLSLDR

Mouse MRNASGPRGP SLATLLFLLL IPEGGCERII GGDTVVPHSR PYMALLKLSS

Human KTICAGALIA KDWVLTAAHC NLNKRSQVIL GAHSITREEP TKQIMLVKKE

Mouse NTICAGALIE KNWVLTAAHC NVGKRSKFIL GAHSINK-EP EQQILTVKKA

#

Human FPYPCYDPAT REGDLKLLQL TEKAKINKYV TILHLPKKGD DVKPGTMCQV

Mouse FPYPCYDETT REGDLQLVRL KKKATVNRNV AILHLPKKGD DVKPGTRCRV

#

Human AGWGRTHNSA SWSDTLREVN ITIIDRKVCN DRNHYNFNPV IGMNMVCAGS

Mouse AGWGRFGNKS APSETLREVN ITVIDRKICN DEKHYNFHPV IGLNMICAGD

Human LRGGRDSCNG DSGSPLLCEG VFRGVTSFGL ENKCGDPRGP GVYILLSKKH

Mouse LRGGKDSCNG DSGSPLLCDG ILRGITSFG- GEKCGDRRWP GVYTFLSDKH

# * *

Human LNWIIMTIKG AV

Mouse 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

H57

D102

R99

P2

P1

S195

P3

P4

D189





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C. S. Craik

Craik Lab Members

Granzyme A

Sandy Waugh

Sami Mahrus

Carly Klein

MT-SP1

Jeonghoon Sun

Ami Bhatt

R. J. Fletterick

Fletterick Lab Members

Mary Jane Budny

and all those helpful people who...

ALS 8.3.1

James Holton

NIH: The $$$ people


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