Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-A resolution - PubMed (original) (raw)

Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-A resolution

T Stams et al. Proc Natl Acad Sci U S A. 1996.

Abstract

It has recently been demonstrated that the C-terminal deletion mutant of recombinant human carbonic anhydrase IV (G267X CA IV) converts the normally glycosylphosphatidylinositol-anchored enzyme into a soluble secretory form which has the same catalytic properties as the membrane-associated enzyme purified from human tissues. We have determined the three-dimensional structure of the secretory form of human CA IV by x-ray crystallographic methods to a resolution of 2.8 A. Although the zinc binding site and the hydrophobic substrate binding pocket of CA IV are generally similar to those of other mammalian isozymes, unique structural differences are found elsewhere in the active site. Two disufide linkages, Cys-6-Cys-11G and Cys-23-Cys-203, stabilize the conformation of the N-terminal domain. The latter disulfide additionally stabilizes an active site loop containing a cis-peptide linkage between Pro-201 and Thr-202 (this loop contains catalytic residue Thr-199). On the opposite side of the active site, the Val-131-Asp-136 segment adopts an extended loop conformation instead of an alpha-helix conformation as found in other isozymes. Finally, the C terminus is surrounded by a substantial electropositive surface potential, which is likely to stabilize the interaction of CA IV with the negatively charged phospholipid headgroups of the membrane. These structural features are unique to CA IV and provide a framework for the design of sulfonamide inhibitors selective for this particular isozyme.

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Figures

Figure 1

Figure 1

Sequence alignment of CA IV and CA II based on superposition of their three-dimensional structures. Relative to CA II, CA IV contains 8 insertions and 3 deletions; the overall sequence identity is 33%. The CA IV numbering scheme used in this work is based on this alignment; inserted residues are indicated by the number of the residue preceding the insertion and the suffix A, B, C, etc.

Figure 2

Figure 2

Omit map of the zinc binding site of CA IV contoured at 4σ; refined atomic coordinates are superimposed and selected active site residues are labeled. Zinc is tetrahedrally coordinated by His-94, His-96, His-119, and a sulfate oxygen. The hydrophobic substrate binding pocket is visible in the upper right background.

Figure 3

Figure 3

Omit map showing the Cys-23–Cys-203 disulfide linkage and the Pro-201–Thr-202 _cis_-peptide linkage (contoured at 4σ). Refined atomic coordinates are superimposed and selected active site residues are labeled.

Figure 4

Figure 4

Least-squares Cα superposition of CA IV (thick bonds) and CA II (thin bonds). The active site zinc ion appears as a gray sphere, and disulfide linkages of CA IV are indicated by large black spheres. Note that although the overall folds of the two isozymes are generally similar, there are significant differences in the region of residue 131 (small black sphere). The extended conformation of the Arg-129–Asn-130 linkage in CA IV may enhance its susceptibility to proteolysis (17, 23).

Figure 5

Figure 5

Electrostatic surface potential of CA IV calculated with

grasp

(55); the color scale ranges from −8_kT_ (red) to +8 kT (blue). Note the extensive positive electrostatic surface potential surrounding the C terminus. This feature reflects the adaptation of the carbonic anhydrase framework for GPI-anchoring and association with the membrane.

Figure 6

Figure 6

Cartoon of the CA IV–membrane interaction. The CA IV isozyme is anchored to the membrane by a GPI tail attached to its C terminus (yellow), which orients the enzyme active site toward the lumen for catalysis. This orientation is further stabilized by the interactions of 11 arginine, lysine, and histidine residues flanking the C terminus with the negatively charged phospholipid headgroups (red) of the membrane. The active site zinc ion appears as a white sphere, and the two disulfide linkages are indicated by bonded yellow spheres. The figure was prepared with

molscript

(56) and

raster3D

(57, 58).

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