Two amino acid substitutions convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase

Abstract

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. Based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.

Figure 1

Sequence alignment of RetGC-1 and ACII C2 in catalytic domain. Amino acid residues conserved in all GCs or all ACs are denoted in gray. Residues conserved between the two types of cyclases are indicated with a “+”. Boxed are amino acids that approach the purine rings in the AC and GC models.

Figure 2

Models of five residues in specificity-determining portion of active site of RetGC-1 (Left) and ACI (Right).

Figure 3

GC and AC activities of wild-type and mutant RetGC-1 constructs. Membranes from transient transfections showing equivalent levels of expression of constructs (a Inset) were assayed for GC activity for 12 min (a) or AC activity for 30 min (b). Stimulated activity was measured in the presence of 4.1 μM GCAP-2. The changed residues in the mutant constructs are indicated in boldface and are underlined.

Figure 4

Determination of Michaelis constants (K_m) of altered specificity mutants. The activities of K/QYDIW, K/QYDLF, and K/RYDLF with increasing amounts of ATP were measured in the presence of 4.1 μM GCAP-2. A curve was fit to each plot by using the Hill equation v = [ATP]ⁿ V_max/K_mⁿ + [ATP]ⁿ. K_m [ATP] values derived from the curve fit were 5.9 ± 1.6 mM for K/QYDLF, 4.5 ± 0.35 for K/QYDIW, and 7.7 ± 1.5 for K/RYDLF. In an independent set of experiments (not shown), we obtained comparable but slightly lower K_m values of 3.6, 3.9, and 3.3 mM [ATP]. The Hill constants obtained from the curve fit were 0.89 ± 0.07, 1.19 ± 0.06, and 1.03 ± 0.07 for the respective mutants, which is similar to the noncooperative behavior we observe with wild-type RetGC-1.

Figure 5

Hypothesis for purine selectivity by guanylyl and adenylyl cyclases. Adapted from Liu et al. (10) and revised following Tesmer et al. (16) and a key role for the E925K mutant. The “A” or “B” after the RetGC-1 amino acid designation represents the polypeptide chain of the dimer.