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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.

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slide1

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

FunctionProtease

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

slide2

What do proteases do?

H

O

H

+

H2O

+

+3HN

C

C

N

C

COO-

R1

H

R2

H

+3HN

C

COO-

R1

H

+3HN

C

COO-

R2

DGo for the rxn is -2kcal/mol

But…

Catalyzed rxn (chymotrypsin) at neutral pH, 37°C: 100/sec

Uncatalyzed rxn at neutral pH, 37°C: 1 X 10-10 /sec

Conditions for chemically catalyzed reaction:

24hrs. @ 6M HCl, 110°C

Koshland, D. (1996) J. Cell. Comp. Phys. Suppl. 1 43:217.

slide3

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+

slide4

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

Cysteine 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

Metalloproteases Zn2+, E270 o-phenanthroline carboxypeptidase digestion

Zn2+, Ca2+ o-phenanthroline thermolysin digestion

E143, H231

slide5

Serine Protease Mechanism – The players

Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

slide7

Serine Protease Mechanism – Oxyanion Hole

Adapted from Voet and Voet (1995) Biochemistry, 2nd ed. John Wiley and Sons, Inc. New York.

slide16

Serine inhibitors

CH3

O

O

S

NH

CH

C

CH2

Cl

O

CH2

Peptide bond mimic

Chloro-methyl ketone [CMK]

TPCK

(L-1-Chloro-3-[4-tosylamido]-4-phenyl-2-butanone)

slide17

Serine inhibitors

CH3

F

CH3

CH

O

P

O

CH

O

CH3

CH3

DFP

Diisopropyl fluorophosphate

slide18

Divergent vs. Convergent Evolution

Catalytic Triad Conserved

Trypsin

Elastase

Subtilisin

Same Fold

slide19

Serpins

Serine protease inhibitors

Irreversible

Disruption of 3º structure

slide20

Ecotin

Serine Protease Inhibitor

Unknown function

Dimeric

1° and 2° binding sites

Cleaved

slide21

Cysteine protease mechanism

159

25

S

N:

N

H

H

O

HN

159

25

P1

+

H

N

N

S

H

O-

O

H

P1

159

25

+

N

H

N

S

H

O-

159

HN

25

P1

N:

N

S

H

O

NH2

P1

Michaelis Complex

Tetrahedral intermediate I

NH2

Tetrahedral intermediate II

H2O

Acyl Intermediate

slide22

Cysteine protease mechanism

159

25

S

N:

N

H

H

O

HN

159

25

P1

+

H

N

N

S

H

O-

O

H

P1

159

25

+

N

H

N

S

H

O-

159

HN

25

P1

N:

N

S

H

O

NH2

P1

Michaelis Complex

Tetrahedral intermediate I

Covalent Intermediate

No Asp102 equivalent

NH2

Tetrahedral intermediate II

H2O

Acyl Intermediate

slide23

Cysteine protease inhibitors

159

25

S

N:

N

H

H

I

O

CH2

C

OH

Iodoacetic acid

E-64

(2S,3S)-3-(N-(1S)-1-[N-(4guanidinobutyl)carbamoyl]3methylbutyl)carbamoyl) oxirane-2-carboxylic acid

slide24

Cystatin Superfamily

Cysteine protease inhibitors

Non-canonical binding

slide25

Acid protease mechanism

P1’

P1

N

O

H

P1

P1’

H

N

O

O

O

H

-O

-

O

H

H

O

O

O

Asp25

-

P1

H

H

O

O

O

P1’

P1

P1’

H

H

O

N

N

H

O-

H

H

O

H

O

O

O

O

O

O-

O

-

-

H

H

Michaelis complex

H

O

O

Asp25’

Asp25

Asp25’

Tetrahedral intermediate

Asp25

Asp25’

Asp25

Asp25’

slide26

Acid protease mechanism

P1

P1’

P1’

P1

H

N

N

O

O

O

H

H

O

O

H

H

O

H

O

O

O

-O

O

-

-

P1

H

H

O

O

O

P1’

P1

P1’

H

H

O

N

N

H

O-

H

H

O

H

O

O

O

O

O

O-

O

-

-

H

H

Michaelis complex

Asp25

Asp25’

Asp25

Asp25’

No covalent intermediate

Activated water

Tetrahedral intermediate

Asp25

Asp25’

Asp25

Asp25’

slide27

Acid protease inhibitors

Indinavir, Roche

slide28

CH3

O

HN

HIV Protease Substrate

NHR’

RHN

N

O

O

OH

Reiling, K. K. et al. Biochemistry (2002) 41:4582-94.

slide29

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!

slide30

Pepsin

HIV Protease

slide31

Metallo protease mechanism

Glu

H

-

His

O

O

His

Zn2+

His

Glu

O

H

Glu

His

H

O

Zn2+

His

O

Zn2+

H

O

O

O

N

H

-O

-O

His

P1

P1’

-

-

His

His

Glu

His

His

Glu

Zn2+

-

O

O

H

Zn2+

O

O

O

P1

O-

H

P1

His

N

His

Glu

P1’

Zn2+

O

O

P1

H

H

N

P1’

slide32

Metallo protease mechanism

Glu

H

-

His

O

O

His

Zn2+

His

Glu

O

H

Glu

His

H

O

Zn2+

His

O

Zn2+

H

O

O

O

N

H

-O

-O

His

P1

P1’

-

-

His

His

Glu

His

His

Glu

Zn2+

-

O

O

H

Zn2+

O

O

O

P1

O-

H

P1

His

N

His

Glu

P1’

Zn2+

O

O

P1

H

H

N

P1’

No covalent intermediate

Activated water

slide33

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

slide34

Thermolysin

Carboxypeptidase A

slide35

Synopsis of Protease Mechanisms

Serine

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

slide36

How Proteases Order Off the Menu

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’

slide38

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]

slide39

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

Harris J. L. et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. PNAS (2000) 97:7754-9.

slide40

P2

O

O

P4

HN

HN

NH

NH

P1

P3

O

O

slide41

Regulation of Proteases – A Few Examples

Trypsinogen

1

1

16

15

Trypsin

16

Zymogens

Pro-peptide that must be cleaved before protease becomes fully active

Enteropeptidase

Zymogen form has distorted oxyanion hole and substrate binding pocket

Compartmentalization

Macromolecular Inhibitors

Host and non-host

slide42

Cytotoxic Lymphocytes

Molecular Biology of the Cell, Garland

slide43

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

slide44

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

slide45

Granzyme Structure

Waugh et al. (2000) Nat. Struct. Biol. 7:762-765

slide47

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

slide49

mOD/min @ 405nm

0

0.05

5

50

[Inhibitor], mM

Macromolecular Inhibition of Granzyme A

Control

mM84R Eco

dM84R Eco

Tryp. Inh.

slide53

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

slide54

Crystallization

Previous 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

slide57

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

slide62

Native Human GrA

Human Mouse

P2 N F

P3 G/A/S F/Y

P4 V/L G

slide63

H -> M GrA

Human Mouse

P2 N F

P3 G/A/S F/Y

P4 V/L G

slide64

Conclusions: Mutational Studies

The residues identified from the model of mouse granzyme A [DL201, 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.

slide65

C. S. Craik

Craik Lab Members

Granzyme A

Sandy Waugh

Sami Mahrus

Carly Klein

MT-SP1

Jeonghoon Sun

Ami Bhatt

The Chemists

Amy Barrios

Alan Marnett

R. J. Fletterick

Fletterick Lab Members

ALS 8.3.1

James Holton

NIH: The $$$ people