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