Biology subjects

Lavriha, P.

Publications and source records attributed to Lavriha, P..

7 recordsLinked to original sources

bioRxiv2026

Neuronal calcium sensors emerge as novel adenylyl cyclase 8 regulators

Adenylyl cyclase 8 (AC8) is a Ca2+-sensitive membrane adenylyl cyclase highly expressed in the central nervous system. AC8-mediated intracellular cyclic adenosine monophosphate (cAMP) accumulation shapes synaptic function, plasticity, and memory formation and is tightly controlled by intracellular Ca2+ and G protein-coupled receptor signaling. Although it is well known that the effects of Ca2+ on AC8 activity are directly mediated by calmodulin, it has remained unclear until now whether other Ca2+-binding proteins also regulate AC8 function. Here, we identify the neuronal calcium sensor hippocalcin-like protein 1 (HPCAL1) as a direct interaction partner of AC8. Using biochemistry, structural proteomics, integrative modeling, cryo-EM and cell-based FRET approaches, we show that HPCAL1 associates with AC8 in a Ca2+-dependent manner. HPCAL1 shows weak positive modulation of AC8 activity in vitro, with sub micromolar affinity. HPCAL1 interacts with flexible regulatory regions of AC8, including the C1b domain and the N- and C-terminal regions. Interestingly, other members of the neuronal calcium sensor family can bind AC8 via the same sites. Together, our study reveals a previously unrecognized mode of Ca2+-dependent AC8 recognition by a group of neuronal calcium sensor proteins, which may be relevant to context-dependent regulation of cAMP signaling.

biochemistry↗

bioRxiv2025

Lipid dependence of connexin-32 gap junction channel conformations

Connexin gap junction channels (GJCs) are a family of proteins that connect the cytoplasms of two neighbouring cells and allow direct intercellular exchange of solutes smaller than [~]1 kDa, ensuring metabolic and electrical coupling of tissues. Connexin-32 (Cx32) GJCs mediate intercellular coupling in a variety of tissues, and specifically in the myelinating Schwann cells. Mutations in Cx32, such as W3S, are associated with peripheral neuropathy, X-linked Charcot-Marie-Tooth (CMT1X) disease. Phospholipids and sterols have been shown to regulate permeation through Cx32 GJCs, although the mechanism of regulation of Cx32 and other GJCs by lipids remains poorly understood. Here, we determine the cryo-EM structures of Cx32 GJCs reconstituted in nanodiscs, revealing that phospholipids block the pore of Cx32 GJC by binding to the site formed by N-terminal gating helices. The phospholipid-bound state is contingent on the presence of a sterol molecule bound to a hydrophobic pocket formed by the N-terminus: the N-terminal helix of Cx32 fails to sustain a phospholipid binding site in the absence of cholesterol hemisuccinate. Consistent with this, the CMT1X-linked W3S mutant which has an impaired sterol binding site adopts a conformation of the N-terminus incompatible with phospholipid binding. Our results indicate that different lipid species control connexin channel gating directly by influencing the conformation of the N-terminal gating helix. One-Sentence SummaryLipids modulate the gating helix conformation in connexin-32 channels

biophysics↗

bioRxiv2023

Mechanism of connexin channel inhibition by mefloquine and 2-aminoethoxydiphenyl borate

Gap junction intercellular communication (GJIC) between two adjacent cells involves direct exchange of cytosolic ions and small molecules via connexin gap junction channels (GJCs). Connexin GJCs have emerged as drug targets, with small molecule connexin inhibitors considered a viable therapeutic strategy in several diseases. The molecular mechanisms of GJC inhibition by known small molecule connexin inhibitors remain unknown, preventing the development of more potent and connexin-specific therapeutics. Here we show that two GJC inhibitors, mefloquine (MFQ) and 2-aminoethoxydiphenyl borate (2APB) bind to Cx32 and block dye permeation across Cx32 hemichannels (HCs) and GJCs. Cryo-EM analysis shows that 2APB binds to "site A", close to the N-terminal gating helix of Cx32 GJC, restricting the entrance to the channel pore. In contrast, MFQ binds to a distinct "site M", deeply buried within the pore. MFQ binding to this site modifies the electrostatic properties of Cx32 pore. Mutagenesis of V37, a key residue located in the site M, renders Cx32 HCs and GJCs insensitive to MFQ-mediated inhibition. Moreover, our cryo-EM analysis, mutagenesis and activity assays show that MFQ targets the M site in Cx43 GJC similarly to Cx32. Taken together, our results point to a conserved inhibitor binding site in connexin channels, opening a new route for development of specific drugs targeting connexins. One-Sentence SummaryInhibition of Cx32 and Cx43 channels via functionally relevant drug binding sites

biochemistry↗

bioRxiv2023

Structural basis of connexin-36 gap junction channel inhibition

Connexin gap junction channels and hemichannels play important roles in intercellular communication and signaling. Some of connexin isoforms are associated with diseases, including hereditary neuropathies, heart disease and cancer. Although small molecule inhibitors of connexins show promise as therapeutic agents, the molecular mechanisms of connexin channel inhibition are unknown. Here, we report the cryo-EM structure of connexin-36 (Cx36) bound to an anti-malarial drug mefloquine at 2.1 [A] resolution. Six drug binding sites partially occlude the pore of each connexon forming the channel. Each drug molecule in the ring makes contacts with residues in the pore-lining pocket and with the neighbouring mefloquine molecules, partially occluding the pore and modifying the pore electrostatics, ultimately reducing solute translocation through the channel. Structures of Cx36 in the presence of quinine and quinidine show a similar mode of drug binding. Molecular dynamics simulations of Cx36 bound to mefloquine show that drug binding affects the kinetics of ion passage through the pore. This previously undescribed mode of connexin channel inhibition presents an opportunity for designing subtype-specific connexin inhibitors. One-sentence summaryMechanism of connexin channel inhibition by small molecules

biophysics↗

bioRxiv2023

Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels

In myelinating Schwann cells, communication between myelin layers is mediated by gap junction channels (GJC) formed by docked connexin 32 hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy with no cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high resolution cryo-EM structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: in the W3S and R22G mutant HCs, the N-terminal helix partially occludes the pore, consistent with an impaired HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X. One-Sentence SummaryConnexin 32 channel structures reveal a gating helix defect in CMT1X disease-associated mutant hemichannels

biophysics↗

bioRxiv2022

Structure of the connexin-43 gap junction channel reveals a closed sieve-like molecular gate

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighboring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease 1-3, yet the structural basis of Cx43 function and regulation has not been determined until now. Here we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 [A] resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and hemichannel cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.

biochemistry↗

bioRxiv2021

Structural basis of adenylyl cyclase 9 activation

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPS, (iv) bound to Gs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation. One Sentence SummaryCryo-EM reveals activator-induced conformational changes in adenylyl cyclase AC9

biochemistry↗