Crystal Structure And Functional Studies Of Scaffold Protein MreC And Hyperthermophilic Archaeal Cas 7 Protein

The vast majority of prokaryotes have cell walls that support and protect cells.They are a natural barrier to protect cells.In eubacteria,the main component of the cell wall is peptidoglycan.Among peptidoglycan,the single-transmembrane protein MreC acts as a central hub in the regulation of peptidoglycan synthesis and cell wall structure,linking the bacterial cytoskeleton with various cell wall modifying enzymes.As a natural immune system of prokaryotes such as bacteria and archaea,the CRISPR systems can be used to fight against invading foreign genetic materials,and they also play a role in protecting cells.Cas 7 family proteins are important components of the CRISPR-Cas systems,and they form the backbone of large interference complexes.This dissertation mainly includes these two research projects.One is to study the structure and function of MreC,a scaffold protein of peptidoglycan on the cell wall of Escherichia coli,and the other is to study the structure and function of Cas 7 from Pyrococcus furiosus Type I-A CRISPR immune system.Peptidoglycan（PG） is an important component of most bacterial cell walls and plays an important role in maintaining bacterial shape,growth,division and resistance to osmotic pressure.The synthesis of peptidoglycan requires not only the participation of various enzymes,but also some scaffold proteins to help the positioning of these enzymes.MreC is considered a scaffold protein that determines cell shape by interacting with proteins involved in peptidoglycan synthesis,acting as a hub in complex formation.Given the critical role of MreC in the formation and maintenance of cell shape,in this paper,we solved the crystal structure of the cell shape-determining protein MreC’s major periplasmic domain from Escherichia coli at2.1?resolution.The overall crystal structure shows an asymmetric unit in a dimer state.Each monomer in the dimer contains a long N-terminal coiled coil and two six-chain barrel domains.The two N-terminal coiled coils in the dimer interact with each other,with the coil of one monomer interposed in the position between the C-terminalβ-barrel and the N-terminal coil of the other monomer.Furthermore,the formation of MreC dimers is associated with the interaction of two N-terminal coiled coils,which contain leucine repeats that are conserved across species,suggesting that MreC dimers are important for their function.Further analysis of the conserved leucine repeats revealed that leucine residues including Leu⁸⁰,Leu⁸⁴,Leu⁹¹,Leu⁹⁴,Leu¹⁰⁴and Leu¹⁰⁸established a leucine zipper that maintained the interaction of the two coiled coils.In addition,there are polar residues that provide hydrogen bonding interactions,which are relatively conserved in bacteria.Although MreCs in different species have lower sequence identity,we speculate that MreCs in other species also form dimers through coiled-coil interactions.Comparison and molecular dynamics simulations of MreC structures from different species suggest that the small interface of theβ-barrel domain facilitates the relative movement of the two monomers.This relative movement can facilitate polymer formation and binding of the remaining related proteins by modulating the relative positions of theβ-barrel domains.In addition,MreC forms antiparallel fibrous structures along the helical direction,which is different from the helical structure of Pseudomonas aeruginosa MreC tetramers.Our structure elucidated the structural basis of the dimerization of E.coli MreC and further elucidated the structural basis of MreC to form antiparallel fibrillar structures along the helical direction,which will provide new scaffolding proteins during peptidoglycan synthesis structural insights and further understanding of MreC polymer formation.As an innate immune system,the CRISPR system is widely distributed in bacteria and archaea.It provides bacteria and archaea with acquired immunity against bacteriophages,viruses,plasmids,and can be used against invading foreign genetic material.There are generally a series of cas genes near the CRISPR sequence,which play an important role in the acquired immune function of the CRISPR system.Cas 7protein is an important part of the CRISPR-Cas system,and it is the core part of the CRISPR-Cas system type I,type III,type IV interference mechanism.The weak sequence homology among the Cas 7 families makes structural data essential for a complete understanding of their structural and functional relationships.Pyrococcus furiosus has three distinct CRISPR-Cas interference systems:Csa（type I-A）,Cst（type I-G）,and Cmr（type III-B）.The type III Cmr system is currently the most studied,and the function of the Pyrococcus furiosus Cmr system against RNA targets has been confirmed in vivo and in vitro.Some studies have confirmed that the Pyrococcus furiosus Cst and Csa effector complexes are also functional.However,the reported Pyrococcus furiosus Cas 7 protein structures are all Cmr-type structures,and there are no other two types of Cas 7 protein structures.Research on Cst and Csa needs to be strengthened.Based on this,we took Csa（type I-A）Cas 7（Csa 2 protein）protein as the research object,and carried out research on the structure and function of Cas 7 protein.In this research,the crystal structure of Cas 7 protein of Pyrococcus furiosus Csa（type I-A）was obtained for the first time by X-ray crystallography.The crystal structure shows that the entire structure of Pyrococcus furiosus Cas 7 protein is crescent-shaped,composed of 14β-sheets and 9α-helices,which can be divided into four domains.The four domains are:LID domain,core domain,C-terminal domain,and N-terminal domain.The core domain has an RRM-like region for RNA recognition.The RRM-like region is a ferredoxin-like fold that is frequently used for RNA recognition.Further structural analysis showed that the core domain of Pyrococcus furiosus Cas 7 evolved from the RRM,so the recognition of RNA by Pyrococcus furiosus Cas 7 may differ in detail from other proteins containing RRM structures in the current protein database.Furthermore,to elucidate the conserved sites of Pyrococcus furiosus Cas 7 protein crystals,we aligned the sequences of Cas7/Csa 2 from ten different species.The alignment results showed that their RNA recognition sites were relatively conserved.Further,the homologous structure of Pyrococcus furiosus Cas 7 protein was searched by DALI.From the Match correlation matrix,it was found that the similarity was mainly concentrated in the RRM-like core domain and the part of the region where the core domain was connected.From this point of view,Cas 7,as a complex at the core of the interference mechanism in the CRISPR-CAS system,can recognize and bind cr RNA.Finally,we give a model of Cas 7 and cr RNA binding by molecular docking simulation.Through the analysis of the structure and function of Cas 7 protein,we provide theoretical support for the elaboration of the cascade of Class 1 Type I-A of the CRISPR immune system,lay a foundation for the subsequent research on the CRISPR immune system,and deepen our understanding of immune survival under extreme conditions in thermophilic archaea.