Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, F-31077 Toulouse, France; Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, UPS, F-31062 Toulouse, France.
Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, UPS, F-31062 Toulouse, France.
J Mol Biol. 2023 May 15;435(10):168048. doi: 10.1016/j.jmb.2023.168048. Epub 2023 Mar 17.
Knr4/Smi1 proteins are specific to the fungal kingdom and their deletion in the model yeast Saccharomyces cerevisiae and the human pathogen Candida albicans results in hypersensitivity to specific antifungal agents and a wide range of parietal stresses. In S. cerevisiae, Knr4 is located at the crossroads of several signalling pathways, including the conserved cell wall integrity and calcineurin pathways. Knr4 interacts genetically and physically with several protein members of those pathways. Its sequence suggests that it contains large intrinsically disordered regions. Here, a combination of small-angle X-ray scattering (SAXS) and crystallographic analysis led to a comprehensive structural view of Knr4. This experimental work unambiguously showed that Knr4 comprises two large intrinsically disordered regions flanking a central globular domain whose structure has been established. The structured domain is itself interrupted by a disordered loop. Using the CRISPR/Cas9 genome editing technique, strains expressing KNR4 genes deleted from different domains were constructed. The N-terminal domain and the loop are essential for optimal resistance to cell wall-binding stressors. The C-terminal disordered domain, on the other hand, acts as a negative regulator of this function of Knr4. The identification of molecular recognition features, the possible presence of secondary structure in these disordered domains and the functional importance of the disordered domains revealed here designate these domains as putative interacting spots with partners in either pathway. Targeting these interacting regions is a promising route to the discovery of inhibitory molecules that could increase the susceptibility of pathogens to the antifungals currently in clinical use.
Knr4/Smi1 蛋白是真菌界所特有的，其在模式酵母酿酒酵母和人类病原体白色念珠菌中的缺失会导致对特定抗真菌剂和广泛的壁应激原的超敏反应。在酿酒酵母中，Knr4 位于几个信号通路的交汇点，包括保守的细胞壁完整性和钙调神经磷酸酶途径。Knr4 在遗传和物理上与这些途径的几个蛋白成员相互作用。它的序列表明它含有大的无规卷曲区域。在这里，小角度 X 射线散射 (SAXS) 和晶体学分析的组合导致了 Knr4 的全面结构视图。这项实验工作明确表明，Knr4 由两个大的无规卷曲区域组成，两侧是一个中央球状结构域，其结构已经建立。结构域本身被一个无序环中断。使用 CRISPR/Cas9 基因组编辑技术，构建了表达从不同结构域缺失的 KNR4 基因的菌株。N 端结构域和环对于最佳抵抗细胞壁结合应激原是必需的。另一方面，C 端无序结构域作为 Knr4 此功能的负调节剂。在这里鉴定的分子识别特征、这些无规卷曲结构域中可能存在的二级结构以及无规卷曲结构域的功能重要性，将这些结构域指定为与这两个途径中的伴侣相互作用的潜在部位。针对这些相互作用区域是发现抑制分子的有前途的途径，这些抑制分子可以增加病原体对目前临床使用的抗真菌药物的敏感性。
