Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases

Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1–5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.


5
The HD region is believed to be a critical element in the membrane and soluble ACs and GCs, as 124 this region couples the N-terminal regulatory domains to the catalytic function of these proteins 125 (23)(24)(25). In Cya, the HD extends from the TM6 ( Fig. 2A-B), forming a coiled-coil observed in the 126 structures of homologous proteins, including AC9 ( Fig. 2C) (9) and sGC (23) (Fig. 3). 127 Interestingly, the size difference between the HD helix in Cya and the HD1 and HD2 helices in 128 AC9 leads to an ~90 rotation of the corresponding TM regions, relative to the catalytic domains 129 (Fig. S5A). This may be an indication that the exact structural alignment of the TM domain and 130 the relatively remote catalytic domain may not be a conserved feature of the membrane ACs. 131 Instead, it is likely that the precise TM-HD and HD-catalytic domain coupling plays the key role 132 in the formation and regulation of the catalytic center in the membrane AC, consistent with the 133 function of the HD as a transducer element in the AC structure (26). 134 The resolved portion of the Cya N-terminus (residues V41ARRQR46), rich in positively charged 135 residues, is immediately adjacent to the HD region. The early work on Cya identified the mutations 136 in this region that disrupt the function of the protein (17), suggesting that the intact residues in the 137 N-terminus stabilize the HD. Our structure provides the structural basis for understanding the 138 likely disruptive effects of these mutations. The positively charged residues R43-R44 likely 139 stabilize the negatively charged surface of the HD (Fig. S5B).

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The TM1-5 bundle as a rail for the HD helices. The TM helices 4, 5 and 6 of Cya form an 141 extensive dimer interface within the membrane (Fig. 2D). The dimer interface residues, close to 142 the "core" of the protein, are relatively well conserved among the Cya homologues from 143 Mycobacteria (Fig. S7), with relatively poorly conserved residues in TM1-3. A comparison of the 144 6-TM bundle of Cya with the corresponding regions in the bovine AC9 (TM1-6 and TM7-12) 145 shows that the helices TM1-5 (and TM7-11 for the AC9) form a well defined structural motif (Fig. 146 2D-E). A striking difference between the Cya and AC9 membrane domains is that the TM region 147 that forms the HD helix is swapped in AC9: the TM12 of AC9 occupies the same position as the 148 TM6 of Cya. Similarly, TM6 in AC9 is placed in a corresponding position relative to the TM7-11 149 ( Fig. 2F-G). The TM1-5 bundle in Cya appears to act as a "guide rail" for the TM6/HD helix of 150 Cya, guiding the correct assembly of the HD coiled coil and the catalytic domain of the cyclase 151 (Fig. 3A). This feature is remarkably similar in AC9, with TM1-5 and TM7-11 arranged in a near-152 identical way (Fig. 3B), and with a closely matching HD core (Fig. 3D). 153 The previous experiments in M. intracellulare Cya have shown that the HD and the TM regions 154 of the protein are critically important for the protein's dimerization and functional assembly (17). 155 The lack of the TM region results in failure to form a stable active dimer of M. tuberculosis 156 Rv1625c / Cya, even in the presence of a nucleotide analogue MANT-GTP, judged by the inability 157 of MANT-GTP to induce crystallization of the protein in a dimeric form (Fig. S4). In contrast, the 158 soluble domain of the M. intracellulare Cya is effectively dimerized by MANT-GTP (17). The 159 importance of the TM domain as a factor that promotes correct protein folding is further illustrated 160 by the ability of the isolated Cya SOL construct to form an inactive domain-swapped dimeric 161 assembly (27). It is thus tempting to suggest that the key function of the TM domain in a membrane 162 AC is to guide the assembly of the enzyme in a catalytically competent form.

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This may have important implications for AC regulation. In a related enzyme, the NO-sensing 164 sGC, the heme-containing NO-receptor domain is fused to the HD region in place of the TM 165 regions seen in Cya or in the mammalian AC9 ( Fig. 3E-G). In its inactive form, the sGC displays 166 a conformation where HD helices are bent, with an accompanying substantial unwinding of the 167 helical domain core (Fig. 3E). Comparison of the Cya HD core with that of the sGC HD core 168 highlights this discrepancy (Fig. 3F). In contrast, activation of sGC is accompanied with a large-169 6 scale conformational change, "straightening" the HD (Fig. 3H) and adopting the HD conformation 170 that closely matches that of Cya (Fig. 3H). The position of the "kink" in the HD of sGC 171 approximately corresponds to the membrane-cytosol interface in Cya. Thus, the very distant yet 172 related proteins sGC and Cya (as well as AC9 and other membrane ACs) may be subject to very 173 similar modes of regulation involving changes in the HD, which may result in changes in the 174 catalytic domain of the protein. While in sGC the process is guided by the heme-containing 175 receptor domain, in the membrane ACs this function is likely performed by the TM domain.

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The TM domain of Cya as a putative receptor module. The structure of Cya revealed several 177 prominent cavities in the TM domain of the protein, which may serve a stabilizing or regulatory 178 role (Fig. 4). A negatively charged cleft (site Ex1) is formed at the extracellular interface of the 179 two 6-TM bundles (Fig. 4A, D, E). The negative charge of this pocket is provided by the residues 180 D123, E164 and D170 of each monomer, facing into the cavity (Fig. 4E). This region may be 181 involved in binding of positively charged ions, small molecules, lipids or peptides. The ability of 182 NB4 nanobody to interact with this pocket spuriously indicates that it may also be a site of 183 interaction with a yet unknown natural protein partner. involve substantial conformational rearrangements opening the connection between the two sites.

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Our density map features a small but prominent density in the site Ex1 (Fig. 4E, Fig. S6), which 202 presently can not be assigned to a specific entity, but which could correspond to a bound metal 203 (Na + , Mg 2 , Mn 2 or a yet unknown component co-purified with the protein from E. coli). It is 204 conceivable that disruption of this negatively charged interface may lead to the loss of the rail  Adenylyl cyclase activity assays were performed as described previously (17). In brief, the assay was   The cryo-EM data were obtained at the SCOPEM facility at ETHZ using a 300 kV Titan Krios electron 397 microscope (FEI) equipped with a K3 direct electron detector with a pixel size of 0.33 Å/pix (in super-398 resolution mode), at a defocus range of -0.5 to -3.0 µm. All movies were dose fractionated into 40 frames.

399
The movies for dataset 1 were recorded with a total dose of 54 e-/Å 2 , dataset 2 -with a dose of 47 e-/Å 2 , 400 and for dataset 3 -a dose of 44 e-/Å 2 .

401
All data processing was performed in relion 3.0 (40). All micrographs were motion corrected using 402 motioncorr 1.2.0 (41) and binned two-fold. All micrographs were CTF corrected using Gctf (42). Particles 403 were autopicked using templates from manual picking. In total 1692104 particles were picked for data set 404 1, 1898968 particles for data set 2 and 990286 particles for data set 3. After several rounds of 2D 405 classification, data set 1, 2 and 3 were left with 253789, 1173076 and 741081 particles respectively. 3D

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Classification with four classes was used to further process each dataset, with C1 and C2 symmetry 407 imposed. The particles from the best classes in each data set were chosen and further refined. The 408 extracellular density for NB4 was masked out for all subsequent refinements to generate refined 3D maps 409 at a resolution of 4.37 Å (dataset 1), 4.25 Å (dataset 2) and 4.61 Å (dataset 3) in C2 symmetry. The particles 410 were merged into a single selection and subjected to refinement, ctf refinement and particle polishing, 411 yielding a final refined map of 3.57 Å resolution (C2 symmetry). The same particle selection produced a   Table S2.

493
-Tryptic peptides are not cleaved by PK at all.

494
-Non-tryptic peptides were cleaved by PK on both sides.

496
Depending on the peptide type an increase or decrease in abundance can be interpreted in different ways.

497
A tryptic peptide that decreases in abundance was additionally cleaved by PK, hence it disappears. This 498 likely means that the protein region became more accessible to PK. On the other hand, a tryptic peptide that 499 decreases in abundance can be interpreted as the region becoming less accessible to PK. A semi-tryptic 500 peptide that increases in abundance can be explained as the protein region cleaved by PK becoming more 501 accessible. A semi-tryptic peptide that decreases in abundance can be explained in two different ways: 502 either the protein region became more protected, hence inaccessible to PK, or the protein region became 503 more accessible and the peptide was not detected because of additional PK cleavage sites that were 504 introduced with the conformational change.