Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases
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Evaluation Summary:
This manuscript reports the first full-length structure of membrane-bound adenylyl cyclase from the pathogen Mycobacterium tuberculosis. The structure provides insights into its potential mechanism of action and reveals similarities to its mammalian counterpart. Thus, this paper is of potential interest to a broad audience including the fields of infectious diseases, signaling, and evolutionary biologists.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
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.
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Evaluation Summary:
This manuscript reports the first full-length structure of membrane-bound adenylyl cyclase from the pathogen Mycobacterium tuberculosis. The structure provides insights into its potential mechanism of action and reveals similarities to its mammalian counterpart. Thus, this paper is of potential interest to a broad audience including the fields of infectious diseases, signaling, and evolutionary biologists.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The MS by Mehta et al, reports the first full-length structure of a polytopic membrane adenylyl cyclase from M. tuberculosis (Rv1625c/Cya). The authors used detergent-purified full-length Cya mixed with a stabilizing nanobody and determined the structure using cryo-EM yielding a 3D reconstruction at 3.8 Å resolution. Full-length Cya (443aa) consists of an N-terminal domain followed by 6 transmembrane (TM) helices, and a helical domain (HD) that connects to the catalytic domain. The full-length protein was active as a dimer and not affected by the presence of the nanobody. In contrast, a soluble form consisting of only HD-cat was not active. The structure of the full-length Cya shows a complex with three molecules of the nanobody, two bound symmetrically to the cytosolic domains and a third one bound …
Reviewer #1 (Public Review):
The MS by Mehta et al, reports the first full-length structure of a polytopic membrane adenylyl cyclase from M. tuberculosis (Rv1625c/Cya). The authors used detergent-purified full-length Cya mixed with a stabilizing nanobody and determined the structure using cryo-EM yielding a 3D reconstruction at 3.8 Å resolution. Full-length Cya (443aa) consists of an N-terminal domain followed by 6 transmembrane (TM) helices, and a helical domain (HD) that connects to the catalytic domain. The full-length protein was active as a dimer and not affected by the presence of the nanobody. In contrast, a soluble form consisting of only HD-cat was not active. The structure of the full-length Cya shows a complex with three molecules of the nanobody, two bound symmetrically to the cytosolic domains and a third one bound asymmetrically to the extracellular surface. The density map covered residues 41-428 of the full-length Cya. The overall topology of each monomer in the Cya dimer corresponded to approximately half of the only known structure of the full-length mammalian membrane-bound AC9, previously solved by the same group. The overall conservation among the bacterial and mammalian cyclases is remarkable. The structure unmasked novel pockets (some conserved with AC9), allowing the authors to suggest a function for the TM bundle as an organizer of the HD for efficient communication to the catalytic domain, and more speculative, a role for the TM, via one of the identified pockets, to act as a putative receptor for yet unidentified ligands able to transduce information via the HD towards enzyme activation.
The overall conclusions of this manuscript are well supported by the data, and although some are speculative, they provide a predictive framework for future studies aiming at biochemical validation.
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Reviewer #2 (Public Review):
Mehta et al report a cryo-EM structure of membrane-bound adenylyl cyclase from Mycobacterium tuberculosis. The structure is a dimer, each subunit consisting of a 6TM membrane domain, a helical domain, and a cytoplasmic adenyl cyclase domain. The overall shape of the dimer is similar to the monomeric (12 TM) mammalian adenylyl cyclase AC9, although there is a swap in the position of the helices that link the TM domain to the helical domain.
An intriguing question is what role the TM domains play in membrane-bound adenylyl cyclases. The authors speculate that they may provide binding sites for ligands and describe 4 different pockets (2 extracellular, 2 cytoplasmic). One extracellular pocket contains the density of a metal ion. Mutation of this pocket reduces the activity of the cytoplasmic catalytic domain …
Reviewer #2 (Public Review):
Mehta et al report a cryo-EM structure of membrane-bound adenylyl cyclase from Mycobacterium tuberculosis. The structure is a dimer, each subunit consisting of a 6TM membrane domain, a helical domain, and a cytoplasmic adenyl cyclase domain. The overall shape of the dimer is similar to the monomeric (12 TM) mammalian adenylyl cyclase AC9, although there is a swap in the position of the helices that link the TM domain to the helical domain.
An intriguing question is what role the TM domains play in membrane-bound adenylyl cyclases. The authors speculate that they may provide binding sites for ligands and describe 4 different pockets (2 extracellular, 2 cytoplasmic). One extracellular pocket contains the density of a metal ion. Mutation of this pocket reduces the activity of the cytoplasmic catalytic domain and induces conformational changes in the whole cytoplasmic region. This provides some evidence for changes in the TM domain being propagated to control cytoplasmic catalytic activity.
In summary, the work is an important structure of bacterial membrane-bound adenylyl cyclase. The description of potential binding pockets is interesting in the step towards understanding the role of the membrane domain although as yet the lack of a clear physiological ligand makes this role still an open question.
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