Crystallographic Studies of Two Proteases: the Aminopeptidase from Vibrio proteolyticus (AAP)

and γ-chymotrypsin.

Aaron G. MoulinPetsko/Ringe LabJanuary 15, 2007

Outline

1. Background on dinuclear metallohydrolases and AAP

2. E151 mutants of AAP

3. Synthesis, kinetics and structure of a Tris-based inhibitor of AAP

Bridged Dinuclear Metalloenzyme

Dinuclear Metalloenzymes

• Large class of enzymes with various functions—amidohydrolases, phosphatases, nucleases.

• Involved in many cellular processes—cell-cycle control, protein degradation, carcinogenesis, tissue repair.

• Implicated in many diseases—neural disorders, bacterial and HIV infections, cancers.

Dinuclear metalloenzymes  Enzyme Metal Reference

Aeromonas aminopeptidase Zn2+

 Structure, (1994), 2, 283-291

Streptomyces griceus aminopeptidase Zn2+ J. Mol. Bio.,(1997), 265, 620-636

Bovine Lens aminopeptidase Zn2+ J. Mol. Bio., (1992), 224, 113-140

Proline aminopeptidase Mn2+ Proc. Nat. Acad. Sci., (1998), 95, 3472

Arginase Mn2+ Nature, (1996), 383, 554

DNA Polymerase Zn2+

Mg2+

EMBO J., (1991), 10, 25-33

Alkaline Phosphatase Zn2+ J. Mol. Bio., (1991), 218, 449-464

HIV-1 Reverse Transcriptase Mn2+ Science, (1991), 252, 88-95

P1 Nuclease Zn2+ EMBO J., (1991), 10, 1607-1619

Glutamine Synthetase Mn2+ J. Mol. Bio., (1989), 264, 17681

Phospholipase C Ca2+ Nature, (1989), 338, 357-360

Manganese Catalase Mn2+ JACS, (1992), 114, 5869-5870

Aminopeptidase from Vibrio proteolyticus

• Dizinc metalloprotease.• Bridging water species.• Isolated from organism Vibrio

proteolyticus.• N-terminal exo-peptidase.• Small (291 a.a., 32 kDa),

nonspecific.• Easily crystallized, diffracts to

atomic resolution.• Recently cloned, mutants

available and are easily purified.

O

Glu152

O

N

N

His256

NN

His97

OO

Asp117

O

Asp179

O

Zn2 Zn1

OH(H)

O

Glu151

O

The Active Site of AAP

Zn2 Zn1O

HNO

NH2

I

OH

Tyr225

3.7

2.41.8 2.1

3.0

Zn2 Zn1O

H2N

HN

O

O

O-

2.3 2.3

3.6

1.8 2.2

OH

NH2HO

OH

Zn1Zn2

CH2

OH

Tyr225

3.45

2.132.211.95 2.21

2.70

Inhibitor Structures of AAP

• p-iodo-D-phenylalanine hydroxamate Ki = 0.4 μM

• L-leucine phosphonic acid Ki = 6.7 μM

• Bestatin Ki = 18 nM• Tris > 20 mM

Zn2 Zn1

O

P

H2N

2.2

O

O

3.9

1.9 2.3

2.1

Chevrier et al. Eur. Jour. Biochem. (1996) 237(2):393-8. Stamper et al. Biochemistry 40, 7035 Stamper et al. Biochemistry. 43(30), pp. 9620-8 Desmarais et al. Structure 10, pp. 1063-1072

O

Glu152

O

N

N

His256

NN

His97

OO

Asp117

O

Asp179

O

Zn2 Zn1

OH(H)

O

Glu151

O

The Active Site of AAP

The Role of E151

• The active sites of several dinuclear metallopeptidases have a putative glutamate base.

• Kinetics studies have demonstrated that replacement of E151 in AAP results in kinetically retarded enzymes.

• Data are consistent with E151 being the catalytic general acid-base.

Roles of various active site residues in AAP, CPG2, SAP and

GCP II

AAP CPG2 SAP GCP II Proposed Function

H97 H112 H85 H377 Zn2+ binding

D99 D114 D87 D379 Hydrogen bonding

D117 D141 D97 D387 Zn2+ binding

E151 E175 E131 E424 Catalytic base

E152 E176 E132 E425 Zn2+ binding

D179 E200 D160 D453 Zn2+ binding

H256 H385 H247 H553 Zn2+ binding

Speno et al. Mol. Pharmacol. (1999) 55(1), pp. 179-185

rAAP E151D-AAP E151H-AAP E151Q-AAP E151A-AAP

kcat (min-1) 4280 2.24 2.00 0.17 ND

Km (μM) 16.4 22.9 25.5 18.5 ---

Bzymek & Holz Jour. Biol. Chem. (2004) 279(30), pp. 31018-25Bzymek et al. Biochemistry (2005) 44(36), pp. 12030-40

Stamper, C. et al. Biochem., 40, p. 7035

Proposed Mechanism1

O

Glu152

O

N

N

His256

NN

His97

OO

Asp117

O

Asp179

O

Zn2 Zn1

OH(H)

O

Glu151

O(H)

Leu-Phe

2Zn2 Zn1

OH(H)

NH

O COO-

H2NO

Glu151

O(H)

3

HN

-OOC

H2N

Zn2

O

Zn1

HO

O

Glu151

O(H)

4

HN

-OOC

H2N

Zn2

O

Zn1

O

H

Glu151

O(H)

O

5H2N O

Zn1Zn2

OH

HN

-OOC

Glu151

O(H)

O

Phe

6

OH2N

Zn2

O

Zn1

H2OLeu

Still Some Questions…

• Which way does the substrate bind?

• What are the reasons for the kinetic retardation of E151 mutants?

• What are the roles of the two zinc ions?

Outline

1. Background on dinuclear metallohydrolases and AAP

2. E151 mutants of AAP

3. Synthesis, kinetics and structure of a Tris-based inhibitor of AAP

E151 Recap

• Mutants E151D- and E151H-AAP are functional, while mutant E151A-AAP is inactive.

• Are there any gross structural changes?

• Can any subtleties in the structures explain rate differences, particularly in the active site?

rAAP E151D-AAP E151H-AAP E151Q-AAP E151A-AAP

kcat (min-1) 4280 2.24 2.00 0.17 ND

Km (μM) 16.4 22.9 25.5 18.5 ---

Crystallization of E151 Mutants

• All mutants crystallized from 4.5 M NaCl, 100 mM KSCN, 100 mM HEPES pH 7.2.

• Crystals grow in 3-4 days for E151D and E151H, in 2-4 weeks for E151A.

• All crystals have hexagonal symmetry.

Data StatisticsData collection and refinement statistics.

E151D E151A E151H

Crystal Data

Space Group P6122 P6122 P6122

Unit Cell Parameters (Å) a= 109.23 109.6 109.1

b= 109.23 109.6 109.1

c= 91.42 91.4 98.4

Data Processing

No. reflections, observed 2,102,450 534,226 457,444

Cutoff (I/) 2.0 2.0 2.8

Rmerge (overall) (%) 8.4 9.3 10.5

Completeness, overall (%) 99.2 99.4 98.4

Model Refinement

Resolution range (Å) 50.0-1.14 50.0-1.75 50.0-1.9

R-factor (%) 13.2 17.8 18.0

Rfree (for 10% of reflections; %) 14.3 20.1 20.5

The Structures Agree Well with the Wild-Type Structure

• The global fold is the same for all mutants.

• There are no gross structural changes anywhere in any mutant.

• The main-chain overlap is good.

E151D-AAP E151A-AAP E151H-AAP

RMSD (Å) 0.21 0.24 0.20

Representative Density

Superimpositions of the Active Sites of E151 Mutants with Wild-Type AAP

Images of the Active Site

• E151H-AAP

• E151D-AAP

• E151A-AAP

E151H-AAP Active Site

Chemical Models and Distances

Zn2 Zn1O

H2N O

2.4

3.4

2.1 1.82.8

Zn2 Zn1O

H2N O

2.5

3.4

2.2 1.93.1

Zn2 Zn1O

H2N O

2.21

3.51

2.10 1.982.88

E151D-AAP

E151A-AAP

Zn2 Zn1O

H2N

HN

O

O

O-

2.3 2.3

3.6

1.8 2.2Bestatin

Zn2 Zn1

O

P

H2N

2.2

O

O

3.9

1.9 2.3

2.1

6

OH2N

Zn2

O

Zn1

Observations

• E151D and E151A crystallize in a product complex whereas E151H does not.

• Product complex looks much like predicted from inhibitor complexes and EPR data.

• Bridging oxygen species seems to be negatively charged.

OH2N

Zn2

O

Zn1

Glu151

OH

O

Zn2 Zn1

HO(H)

H2O

H3NO O

Lower the pH• E151D-AAP crystals were

grown and transferred to pH 4.5.

O O

H3N

Zn1 Zn2

O O

Asp117

OH

A Revised Mechanism for AAP1

O

Glu152

O

N

N

His256

NN

His97

OO

Asp117

O

Asp179

O

Zn2 Zn1

OH(H)

O

Glu151

O(H)

3

HN

-OOC

H2N

Zn2

O

Zn1

HO

O

Glu151

O(H)

Leu-Phe

2Zn2 Zn1

OH(H)

NH

O COO-

H2NO

Glu151

O(H)

4

HN

-OOC

H2N

Zn2

O

Zn1

O

H

Glu151

O(H)

O

6

OH2N

Zn2

O

Zn1

Glu151

OH

O

Phe

Leu3a

HN

-OOC

H2N

Zn2

O

Zn1

OH

O

Glu151

O(H)

5

H2N O

Zn1Zn2

OH

HN

-OOC

Glu151

O(H)

O

OH3N

Zn2

O

Zn1

H2O

OH(H)

7

Conclusions

• The E151 mutants are kinetically retarded due to distance/orientation. • The bridging oxygen species of the product complex carries negative

charge.• The product (and likely the substrate as well) binds with the amine

coordinating to Zn2.• Zn2 seems to serve to coordinate the amine, as predicted.• Zn1 is still likely to active the carbonyl.• A terminal water species is observed at Zn2, the opposite of

prediction.• E151 is apparently responsible for a number of proton shuttling

events, including the protonation of the amine of the product.

Outline

• Background on dinuclear metallohydrolases and AAP

• E151 mutants of AAP

• Synthesis, kinetics and structure of a Tris-based inhibitor of AAP

Tris Can Inhibit AAP

• Tris inhibits AAP with Ki > 20 mM

• Inhibits competitively

• Binds to the active site in “backwards” fashion

Desmarais et al.Structure (2002) 10(8):1063-72

Can Tris Be Used as a Scaffold to Design New Inhibitors?

• Substitution of one hydroxyl for a gain in binding energy

• Synthesis should be general

• Should be possible with commercially available starting materials

Desmarais et al.Structure (2002) 10(8):1063-72

The Synthesis Scheme for Tris-Based Inhibitor “Benzyl-Tris”

“Benzyl-Tris” Inhibits AAP Competitively

• Inhibits with calculated Ki = 180 μM.

Crystal Growth and Diffraction Statistics

• Wild-type crystals grown from 4.5 M NaCl, 100 mM KSCN, 100 mM HEPES pH 7.2

• Soaked with “benzyl-tris” at 300 mM

Data collection and refinement statistics.

Crystal Data

Space Group P6122

Unit Cell Parameters (Å) a=110.11b=110.11c=91.23

Data Processing

No. reflections, observed 2340130

Rmerge (overall) (%) 10.1

Completeness, overall (%) 98.9

Model Refinement

Resolution range (Å) 50-1.14

R-factor (%) 12.1

Rfree (for 2053 reflections; %) 14.2

The Active Site Density

Comparison of the Tris and “Benzyl-Tris” Structures

Anisotropic Displacement Paramters for Tris and “Benzyl-Tris”

Conclusions

• Tris can be used as scaffold to build inhibitors for AAP, and possibly other dinuclear metalloenzymes.

• A general synthesis has been developed and implemented for several compounds.

• The “benzyl-Tris” inhibitor is competitive with AAP, with a Ki = 180 μM.

• The structure reveals a binding mode analogous to Tris, though one metal-coordinating ligand has been lost.

• Analysis of anisotropic displacement parameters shows that Tris ligand loss had highest thermal motion and likely lower binding energy.

• Loss of the metal-coordinating ligand is compensated by gain of hydrophobic interactions in the binding pocket.

Acknowledgements• Greg• Dagmar

• Tim• Mark• Todd

• Alejandro• Cheryl• Bryan• Gabe• Ed• Wally• Jose• Dali• Nilou• Quyen• Shulin• Rest of the PR Lab• Alex Milshteyn

• Rick Holz• Krzysztof Bzymek

• Rex Pratt• Jason Bell

• BioCARS• SSRL• GM/CA CAT• X25A• X6A

• Mom• Dad• Karen• George & Vera• Dorothy

Fenn T.D. et al. (2003) POVScript. J. Appl. Cryst. 36(2), pp. 944-947.