Strategy for IGPS Structural Modification

Having chosen two enzymes with similar structure, IGPS and PRAI, Altamirano and colleagues began modifying the amino acid sequence of IGPS to confer upon it the activity of PRAI.

Removal of the N-terminal extension
First, the IGPS active site was modified to bind phosphoribosyl anthranilate, the substrate of PRAI. As with PRAI, IGPS possesses an anthranilate moiety binding site at loop b2a2, as well as phosphate binding sites at b7a7 and b8a8. Unlike PRAI, IGPS contains an N-terminal extension of 49 residues, which includes an initial helix attached to the rest of the enzyme via a long loop (Figure 9). This extension was deleted to create IGPS49, an unstable and inactive protein. However, IGPS49 was capable of binding an IGPS inhibitor, 3H-rCdrp, indicating that the necessary binding sites were preserved.

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Figure 9 The image at right is a superimposed structure comparison between PRAI, shown in pale yellow, and IGPS, shown in blue. The 49 residue extension of IGPS is shown in magenta and has no counterpart in PRAI.
The "Gatekeeper" loops
There are two other major structural differences between IGPS and PRAI that involve the active site (Figure 10). The extent of the active site exposure is determined by loops b1a1 and b6a6; in PRAI, these loops partially cover the active site, while in IGPS the active site is even less exposed. In PRAI, b1a1 is only four amino acids long, while the corresponding IGPS loop is 15 residues long. b6a6 in both enzymes is 11 residues long, but has a completely different orientation in each enzyme. Furthermore, the b6a6 loop in all PRAI enzymes (species to species) has a highly conserved sequence.
Figure 10 Again, PRAI (light yellow) has been superimposed on IGPS (blue). Loop b1a1 is shown in red; loop b6a6, in orange. The IGPS extension is depicted in magenta. From this angle, the TIM barrel structure of both enzymes is conspicuous. The binding pocket is in the center of the barrel, behind the b1a1 and b6a6 loops.

Modification of loop b1a1...
In order to make IGPS b1a1 more similar to PRAI b1a1, the original IGPS b1a1 was deleted, and new sequences between four and seven residues long were added to create several new mutants. This was accomplished by inserting a conserved sequence in the b1a1 DNA that contained G and K to mimic the normal PRAI loops. Three different gene libraries of the mutant IGPS DNA (IGPS49L1, IGPS49L1RGD, and IGPS49LSV) were then created, and the mutant genes in each library were expressed, yielding a different IGPS mutant. The mutant with the strongest binding characteristics was selected.

... and b6a6
A similar procedure was carried out for the b6a6 loop, except that the first three libraries were combined to produce one large library, IGPS49L1L6. The resulting mutant protein was an a/b barrel which could bind the desired substrate, though it lacked any chemical activity.

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Nikki Jarrett, 11/20/00
Biochemistry 462bH, Dr. D. Bourque