Manna Tuhin, Dey Subhamoy, Karmakar Monalisha, Panda Amiya Kumar, Ghosh Chandradipa
Deparment of Human Physiology, Vidyasagar University, Midnapore, West Bengal, India.
Centre for Life Sciences, Vidyasagar University, Midnapore, West Bengal, India.
Curr Res Struct Biol. 2025 Apr 9;9:100166. doi: 10.1016/j.crstbi.2025.100166. eCollection 2025 Jun.
During various stages of its life cycle, initiate biofilm signalling cascade. Intercellular high level of the signalling nucleotide 3'-5' cyclic dimeric guanosine monophosphate (c-di-GMP), synthesized by diguanylate cyclases (DGCs) from its precursor molecule GTP, is crucial for biofilm formation. Present study endeavours to approaches in evaluating genomic, physicochemical, topological and functional properties of six c-di-GMP regulatory DGCs (CdgA, CdgH, CdgK, CdgL, CdgM, VpvC) of . Genomic investigations unveiled that codon preferences were inclined towards AU ending over GC ending codons and overall GC content ranged from 44.6 to 49.5 with codon adaptation index ranging from 0.707 to 0.783. Topological analyses deciphered the presence of transmembrane domains in all proteins. All the DGCs were acidic, hydrophilic and thermostable. Only CdgA, CdgH and VpvC were predicted to be stable during conditions. Non-polar amino acids with leucine being the most abundant amino acid among these DGCs with α-helix as the predominant secondary structure, responsible for forming the transmembrane regions by secondary structure analysis. Tertiary structures of the proteins were obtained by computation using AlphaFold and trRosetta. Predicted structures by both the servers were compared in various aspects using PROCHECK, ERRAT and Modfold8 servers. Selected 3D structures were refined using GalaxyRefine. InterPro Scan revealed presence of a conserved GGDEF domain in all DGCs and predicted the active site residues in the GGDEF domain. Molecular docking studies using CB-DOCK 2 tool revealed that among the DGCs, VpvC exhibited highest affinity for GTP (-5.6 kcal/mol), which was closely followed by CdgL (-5.5 kcal/mol). MD simulations depicted all DGC-GTP complexes to be stable due to its considerably low eigenvalues. Such studies are considered to provide maiden insights into the genomic and structural properties of DGCs, actively involved in biofilm signalling systems, and it is projected to be beneficial in the discovery of novel DGC inhibitors that can target and downregulate the c-di-GMP regulatory system to develop anti-biofilm strategies against the cholera pathogen.
在其生命周期的各个阶段，启动生物膜信号级联反应。细胞间高水平的信号核苷酸3'-5'环二聚鸟苷单磷酸（c-di-GMP）由双鸟苷酸环化酶（DGCs）从其前体分子鸟苷三磷酸（GTP）合成，对生物膜形成至关重要。本研究致力于评估[具体对象]的六种c-di-GMP调节性DGCs（CdgA、CdgH、CdgK、CdgL、CdgM、VpvC）的基因组、物理化学、拓扑和功能特性的方法。基因组研究表明，密码子偏好倾向于以AU结尾而非GC结尾的密码子，总体GC含量在44.6至49.5之间，密码子适应指数在0.707至0.783之间。拓扑分析表明所有蛋白质中都存在跨膜结构域。所有DGCs都是酸性、亲水性且热稳定的。仅预测CdgA、CdgH和VpvC在[具体条件]下是稳定的。这些DGCs中以亮氨酸为主的非极性氨基酸最为丰富，通过二级结构分析，α-螺旋是主要二级结构，负责形成跨膜区域。蛋白质的三级结构通过使用AlphaFold和trRosetta进行计算获得。使用PROCHECK、ERRAT和Modfold8服务器在各个方面比较了两个服务器预测的结构。使用GalaxyRefine对选定的3D结构进行了优化。InterPro Scan显示所有DGCs中都存在保守的GGDEF结构域，并预测了GGDEF结构域中的活性位点残基。使用CB-DOCK 2工具进行分子对接研究表明，在DGCs中，VpvC对GTP表现出最高亲和力（-5.6千卡/摩尔），其次是CdgL（-5.5千卡/摩尔）。分子动力学模拟表明，由于其相当低的特征值，所有DGC-GTP复合物都是稳定的。此类研究被认为为积极参与生物膜信号系统的DGCs的基因组和结构特性提供了初步见解，并预计有利于发现新型DGC抑制剂，这些抑制剂可以靶向并下调c-di-GMP调节系统，以制定针对霍乱病原体的抗生物膜策略。
