BIOCHEMICAL

Vol. 181, No. 2, 1991 December

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 579-584

16, 1991

Structural

Properties Satish

of Human Carbonic Anhydrase K. Nair and David

II at pH 9.5

W. Christianson*

Department of Chemistry University of Pennsylvania Philadelphia, Pennsylvania 191046323

Received

October

3, 1991

The structure of human carbonic anhydrase II at pH 9.5 has been studied by X-ray crystallographic methods to 2.2 di resolution. These studies complement those pecformed under acidic conditions in which the catalytically-important proton-shuttle group, His-64, exhibits conformational mobility about side-chain torsion angle xi. However, no structural changes are observed in the conformation of His-64 at high pH. Therefore, we conclude that the protonation of His-64 (as well as zinc-bound hydroxide) may be a factor which contributes to the predominantly “out” conformation for His-64 observed at low pH. 0 1991 Academic Press, Inc.

The (CAD)

crystal

reveals

structure

a roughly-spherical

a catalytically-required

three histidine

residues

solvent

In addition

in the function

hydroxide

for His-64.

structure

surrounding

(i.e. rotations

the Thr-2OO+Ser

mutant

by 104” about

substitution

*

His-96 and His-119),

polyhedron.

crystallographic

mobility

ring-flipping

tates,

At physiological

anhydrase of which

oc-

pH, zinc is coordinated

by

and hydroxide

His-64,

(about

8 A away) through

studies

of CA11 indicate

resides

COs hydration

ion completes

to the zinc ligands,

of CAD

II

another

a

active

a catalytic

proton

shuttle,

a hydrogen

bonded

solvent

(3, 4). X-ray

tional

control.

(His-94,

is implicated

engages zinc-bound network

carbonic

active site cavity, at the bottom

of diffusion

coordination

site histidine

human

zinc ion (1, 2): it is here that catalytic

curs near the limit

tetrahedral

of the metalloenzyme

xi,

at position

To whom correspondence

In the native

structure

His-64 is consistent about of CAII

200 (5).

at pH 8.0 reveals

should

I n addition,

of blood

with

side chain torsion

as a compensatory

a high

response

degree CAII

of conformaat pH 8.5, the

an interpretation angle xZ) (2). that

of imidazole The structure

the His-64

to the conservative

structures

of

side chain amino

of CA11 determined

roacid

under

be addressed. 0006-291x/91 579

All

$1.50

Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.

Vol.

181, No. 2, 1991

acidic

conditions

by differences xr in order

BIOCHEMICAL

show that

His-64

(7), a detailed

a range of conformations

characterized

angle xi

values (6).

rotates

the binding

understanding

will assist rational In order

of factors

the structure

to a resolution

Since His-64

of thienothiopyran-2-sulfonamide which

influence

behavior

observed

Materials

and

in the enzyme

is fully active

mobility

of glaucoma

under

of CA11 at pH 9.5 by X-ray

of 2.2 A. The enzyme

differences

of His-64

about

inhibitors

t,his conformational

drug design efforts aimed at the treatment to assess the dynamic

have determined

RESEARCH COMMUNICATIONS

occupies

in side chain torsion

to accommodate

AND BIOPHYSlCAL

(8).

basic conditions,

crystallographic

we

methods

at pH 9.5 (9), so any structural

at this pH, if any, must sustain

catalysis.

Methods

harvested, and transHuman carbonic anhydrase II (S i g ma ) was crystallized, ferred to high-pa buffer in a procedure described previously (6, 10). Crystals sitting in a buffer droplet of 1.75 - 2.5 M (NH4)rS04, 3 mM NaNs, 150 mM NaCl, aminomethane hydrochloride), pH 8.0 were 50 mM Tris-HCl (Tris[hydroxymethyl]equilibrated against a buffer solution of 1.75-2.5 M (NH4)rS04, 50 mM CAPS0 j3[Cyclohexylaminol-2-hydroxy-1-propane-sulfonic acid), pH 9.5. The final pH of the buffer droplet in which each crystal was immersed was determined to be 9.5 f 0.2 using high-sensitivity pH paper from ColorpHast (EM Science). All X-ray diffraction data were collected at room temperature X-1OOA multiwire area detector mounted on a Rigaku RU-200 rotating generator. Raw data frames were analyzed using the BUDDHA (11) merged using the software PROTEIN (12). R e 1evant data collection found in Table I.

on a Siemens anode X-ray package and statistics are

The structure of the enzyme at pH 9.5 was refined against structure factors calculated from the refined structure of CAII at pH 8.5 (2) less the atomic coordinates of His-64 and active site water molecules. Model building was performed with the PS390. Crystalgraphics software FRODO (13) installed on an Evans and Sutherland lographic refinement utilized the reciprocal space least-squares algorithm of Konnert for Hisand Hendrickson, PROLSQ (14). A c t ive site water molecules and coordinates 64 were added when the crystallographic R-factor dropped below 0.19. The R-factor for the final model was 0.18, and relevant refinement statistics are recorded in Table I. Coordinates have been deposited in the Brookhaven Protein Data Bank (15).

Results

and

Discussion

The three-dimensional by Eriksson

and colleagues

nates (using

the software

A between culated

CAII

structure

of CA11 at pH 9.5 is similar

at pH 8.5 (2). Least squares superposition INSIGHT

pH 9.5 and 8.5 CAIIs.

for pH 9.5 CAII

the overall

structure

(Biosym,

Inc.))

yields

does not exhibit

basic conditions. 580

derived significant

of Ca coordi-

an rms difference

Given the rms coordinate

from relationships

to that reported

by Luzzati

error

of ca. 0.2 A cal-

(16), it appears

pH-dependent

of 0.2

variation

that under

Vol.

BIOCHEMICAL

181, No. 2, 1991

AND BIOPHYSICAL

Table Data Collection

RESEARCH COMMUNICATIONS

I

and Refinement

Statistics

for

CAD at pH 9.5 2

Number of crystals Number of measured reflections Number of unique reflections Maximum resolution (A) b,(W Number of water molecules in final cycle of refinement R factorb RMS deviation from ideal bond lengths (At RMS deviation from ideal bond angles (degrees) RMS deviation from ideal planarity (A) RMS deviation from ideal cbirality (As)

28,781 10,632 2.2 0.091 110 0.180 0.007 2.0 0.011 0.093

a Rmerge for replicate reflections, R = ~llF~~l~l~~ IF/J = scaled structure factor for reflection h in data set i. = average structure factor for reflection h from replicate data. b Crystallographic R factor, R = ZlIF,I - lFcII/EIF,,I; lFol and IFJ are the observed and calculated structure factors, respectively.

The imidazole this higher density,

side chain

pH and remains

interpretable

determined

Although

“out”

conformer The

position

coordination

which

and is an inhibitor to this ligand. non-protein

zinc-bound

and azide give

zinc ligand

remains

which

or instead

rise to the elongated

favorable

in the predominant

conformer

(Figure

the crystal density

density

1).

was soaked, corresponding

hydrogen

an exclusive

the

However,

corresponding

of disordered,

that equilibrium

occupied

at pH 8.5.

given the weaker

electron

the coordination

tions (6), are not evident

in which

However,

of

at pH 9.5, and

observed

a pair

as an azide anion.

(17); it is quite reasonable

have been observed

of this density

2, it is clear that the elongated as either

is reminiscent

a partially

(17), may affect the electron

to the bonded

interpretation

of

binding

of azide under

populations

of hydroxide

density.

polyhedron

over the pH range of 5.7 - 9.5 as observed Changes

Weak electron

at pH 6.5 (6) and pH 8.0

tetrahedral

is near that

at

of His-64 in structures

as solvent,

the appearance

in the buffer droplet

is interpretable

In conclusion,

conformer

this density

with

azide is not chemically

basic conditions

conformation.

maps calculated

polyhedron

is present

In Figure

molecules,

interpret

of the enzyme

ligand

an alternate

density

is not inconsistent

of the non-protein

azide anion,

water

electron

we tentatively

zinc

in the “in”

changes

lower pH values, persists at pH 9.5 (this density

in difference

(10).

His-64 shows no conformational

predominantly

as solvent and/or

at certain

that observed

of residue

of zinc in CA11 remains

in several crystallographic of His-64

in crystallographic under basic conditions: 581

in its “in”

studies

performed

His-64 remains

*

tetrahedral

investigations. “out”

under

equilibrium, acidic

predominantly

condiin the

Vol.

/

181, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/

>

Gi

Fidure 1. Difference electron density map of pH 9.5 CAD calculated with Fourier coefficients IF,,1 - lFcl and phases derived from the final model, less the atomic coordinates of His-64 and active site solvent molecules. The map is contoured at +2.3a (dashed lines), and refined atomic coordinates of pH 9.5 CA11 are superimposed (thick bonds): for comparison, the refined coordinates of pH 5.7 CA11 are also superimposed (thin bonds). Although a continuous trace of density places the imidazole group of His-64 in the “in” conformation, density interpretable as solvent (OHH 432) may instead represent a partial-occupancy “out” conformer of His-64; the “out” conformer is observed exclusively at pH 5.7.

Figure 2. Difference electron density map of pH 9.5 CA11 calculated with Fourier coefficients IF,1 - IF,1 and phases derived from the final model less the atomic coordinates of the non-protein zinc ligand. The map is contoured at $2.50, and refined atomic coordinates of pH 9.5 CA11 are superimposed. The non-protein zinc-ligand is characterized by elongated density and may reflect an equilibrium between ainchydroxide and zinc-azide species.

582

Vol.

181, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

“in” conformation at pH 9.5. Therefore, we conclude that the protonation of His-64

[PK, = 7.1 (WI, as well aszinc-bound solvent [pK, = ca. 7.0 (19, 20)], may stabilize the “out” conformer of His-64 in X-ray crystallographic studies performed at lower pH values. Given that the “out” conformation of His-64 is observed in the Thr-2OO-+Ser mutant of CA11 at pH 8.0 (5)) a more general conclusion may be that the conformation of His-64 responds to structural changesin its environment: these changes may include protein, solvent, or counterion species. The structural implications of the His-64 conformational equilibrium will no doubt be important for understanding proton transfer in the CA11 active site (3, 4)) as well asfor the designof tight-binding enzyme inhibitors (7).

Acknowledgments

We thank the NSF for grant DIR-8821184 (in support of the X-ray data acquisition equipment) and the NIH for grant GM45614. Additionally, D.W.C. is grateful to the Office of Naval Researchfor a Young Investigator Award. S.K.N. is supported by N.I.H. Training Grant GM07229.

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