Role of the nucleotidyl cyclase helical domain in catalytically active dimer formation

Abstract

Nucleotidyl cyclases, including membrane-integral and soluble adenylyl and guanylyl cyclases, are central components in a wide range of signaling pathways. These proteins are architecturally diverse, yet many of them share a conserved feature, a helical region that precedes the catalytic cyclase domain. The role of this region in cyclase dimerization has been a subject of debate. Although mutations within this region in various cyclases have been linked to genetic diseases, the molecular details of their effects on the enzymes remain unknown. Here, we report an X-ray structure of the cytosolic portion of the membrane-integral adenylyl cyclase Cya from Mycobacterium intracellulare in a nucleotide-bound state. The helical domains of each Cya monomer form a tight hairpin, bringing the two catalytic domains into an active dimerized state. Mutations in the helical domain of Cya mimic the disease-related mutations in human proteins, recapitulating the profiles of the corresponding mutated enzymes, adenylyl cyclase-5 and retinal guanylyl cyclase-1. Our experiments with full-length Cya and its cytosolic domain link the mutations to protein stability, and the ability to induce an active dimeric conformation of the catalytic domains. Sequence conservation indicates that this domain is an integral part of cyclase machinery across protein families and species. Our study provides evidence for a role of the helical domain in establishing a catalytically competent dimeric cyclase conformation. Our results also suggest that the disease-associated mutations in the corresponding regions of human nucleotidyl cyclases disrupt the normal helical domain structure.

Keywords: Cya; X-ray structure; adenylyl cyclase; dimerization; helical domain.

Fig. 1.

Structure of the cytosolic domain of M. intracellulare Cya. (A) A schematic representation of Cya; cytosolic domain used for crystallization in this study is circled. (B) Enzymatic activity of purified full-length Cya and Cya_SOL; values are shown as mean ± SEM (n = 4), the K_m and V_max are shown in SI Appendix, Table S2. Different views of the structure of the soluble portion of Cya are shown. (C) Side view of two asymmetric units of Cya_SOL crystals (space group C 1 2 1); 11 chains assemble into a continuous protein fiber along the long axis of the crystalline lattice; 10 of the monomers are assembled into 5 active dimers (chains A–J), with one (chain K) interacting with its counterpart in a neighbor asymmetric unit. (D) Structure of Cya_SOL reveals two molecules of MANT-GTP ligand per dimer, with two Mn²⁺ ions bound per MANT-GTP. (E) The structure reveals the dimerized catalytic domain (CAT, colored forest and pale green) and the helical domain (HD; colored orange and light orange).

Fig. 2.

EPR spectroscopy confirms the cytosolic domain arrangement in the full-length Cya. (A) A rigid position within the catalytic domain, N328, was chosen for mutation to Cys and spin labeling. (B) CW experiments show that the N328C position was successfully labeled and the position in the context of Cya_ΔCYS (gray) and Cya_SOLΔCys (black) is rather immobile. (C) Intensity-normalized Q-band DEER primary data of Cya_ΔCYS-N328C. Details on acquisition and analysis of DEER data are in SI Appendix. (D) Simulated (dashed line) and experimentally determined distance distributions. The blue curves correspond to the apo state, while the red ones to the MANT-GTP bound; the Cya_SOL structure was used as a template for atom positions during simulations. (E and F) Same as in C and D, with data for the Cya_SOLΔCys-N328C mutant; all observations in C–F are representative of three experiments. Dashed lines in C and E correspond to the decay functions used for background correction. From the crossings of the background decay functions with the normalized intensity axes (y axes) DEER modulation depths are determined (see SI Appendix for details).

Fig. 3.

Cya serves as a model for helical domain mutations implicated in genetic diseases in nucleotidyl cyclases. (A and B) Position of the mutants in the helical domain structure (A) and alignment of Cya with GCE and AC5 (c1 and c2) helical domains (B). Equivalently colored spheres and arrows indicate the positions of the affected mutations that are mapped on Cya_SOL structure in A or in sequence alignment in B. The alignment is shaded according to the BLOSUM62 score. (C) Enzymatic activity of full-length Cya, compared with five different mutants linked to human diseases grafted onto Cya construct. For each construct, the column on the Left, labeled with “−”, indicates the assays performed in the absence of competitor; the column on the Right, labeled “M”, indicates assays performed in the presence of MANT-GTP. Values are shown as mean ± SEM (n = 3–8); statistical significance was determined using one-way ANOVA followed by a Sidak’s test (catalysis rates of M212K and P228S are significantly different from the wild type, with P < 0.005 and P < 0.0001, respectively). (D) CPM-based stability assay of the same purified mutant proteins, in the apo (Left) and in the MANT-GTP–bound (Right) state; values are shown as mean ± SEM (n = 3–6). Statistical significance was assessed using one-way ANOVA followed by a Sidak’s test (all samples, apart from S219A in the MANT-GTP bound state, are significantly different from the wild-type; P < 0.05).

Fig. 4.

The P228S mutation disrupts the enzymatic activity but preserves the nucleotide interactions with the catalytic domains. (A) Ability of the Cya_SOL to produce cAMP is severely impaired by removal of the helical domain or by introduction of a P228S mutation. (B) Fluorescence-based MANT-GTP binding assay confirms that all three constructs are capable of binding the nucleotide analog. Excitation wavelength was 350 nm; emission was recorded in the range of 370–500 nm. Inset sketches describe each construct, colored according to the colors in A. (C) Normalized analytical ultracentrifugation (AUC) profiles of the three constructs; AUC was performed at two protein concentrations, 0.3 mg/mL (open circles) and 1 mg/mL (filled circles), in the absence (blue) or presence of MANT-GTP (red). Cya_SOL was compared with the P228S mutant and the catalytic domain only. (Top) A propensity to dimerize in the absence of nucleotide is evident from a discrete peak in the Cya_SOL sample (asterisk) and only at 1 mg/mL; the appearance of this intermediate peak at a higher protein concentration is indicative of a monomer-dimer equilibrium. (Middle and Bottom) Disruption of the helical domain by truncation or P228S mutation abolishes dimerization. Monomers and dimers are indicated by “m” and “d,” linked by an arrow. For all experiments, values are shown as mean ± SEM (n = 3). (D) Interface between the Cya helical domain and its catalytic site. The surface representation depicts the shape of Cya_SOL dimer. A chain of the Cya_SOL monomer is shown in ribbon representation; MANT-GTP and 2xMn²⁺ ions are represented as spheres; Mn²⁺ ions are represented as blue spheres. The boxed area indicates side chains of P228 in the helical domain (HD) and the loop formed by residues V353-W367, which acts as the interface between the helical domain and the catalytic domain (CAT).

Fig. 5.

Conservation of helical domain core in nucleotidyl cyclases across species and protein families. (A) Cya_SOL dimer shown with one monomer in surface representation and the other shown in ribbon representation. Interface analysis using Eppic (46) reveals strong conservation of interface residues within close homologs of Cya. Sequence identity cutoff was kept at 30%, but similar results were obtained using 15–50% identity cutoff. The color code reflects the sequence entropy, as defined in Eppic: blue, low entropy/high conservation; red, high entropy/low conservation (Eppic database statistics for this search: UniProt_2016_07 and PDB release of 18–10-2016). (B) Map of the core residues (colored in blue) at the Cya_SOL dimer interface, as defined in Eppic (>95% buried surface). The helical domain contributes substantially to the dimer interface residues of Cya_SOL, with 9 of 23 core residues mapped to the helical domain interface. (C) A high conservation of the helical domain core features is revealed by HMMER search (www.hmmer.org) (47) in mammalian proteome database using M. intracellulare Cya cytosolic domain sequence as a search template. The HMM logo was generated from the HMMER multiple sequence alignment output using Skylign (www.skylign.org). The red line underlining the HMM logo indicates the corresponding residues resolved in Cya_SOL structure; blue rectangles indicate core residues (as shown in B). Asterisks indicate residues implicated in genetic diseases upon mutation in retGC1 (detailed in the text). (D) The helical domain is conserved across protein families and architectures and is present in bacterial and mammalian membrane ACs (“tmAC”; adjacent sketches represent mycobacterial Rv1625c/Cya and mammalian AC1–9), receptor GCs (“tmGC”), NO-sensitive soluble GC (“sGC”), and soluble AC10 (“sAC”).