Preliminary Crystallographic Study Of Amuc＿1102 And Amuc＿1098 Protein From Akkermansia Muciniphila

Akkermansia muciniphila is a beneficial microorganism that colonizes the human intestine.It is a gram-negative bacteria and is strictly anaerobic.As the body of beneficial microorganisms,A.muciniphila is closely related to human intestinal health and has a good preventive and protective effect on diseases related to intestinal metabolism.The proteins encoded by the Amuc＿1098-Amuc＿1102 gene cluster in A.muciniphila have a high proportion of its outer membrane components and are highly expressed.Analyzing the protein sequence in the gene cluster,it is found that the gene cluster is considered to be a protein group related to the formation and assembly of type Ⅳ pilus(T4P).However,the function of the protein encoded by Amuc＿1102 in this gene cluster cannot be determined by sequence analysis,and its function is not yet known.Therefore,the main object of this research is the Amuc＿1102 protein.By analyzing the three-dimensional structure of the protein and further analysis of the folding mode,the possible function of Amuc＿1102 can be inferred.In this project,Amuc＿1102 recombinant protein was obtained by heterologous expression in E.coli,and purified protein by Nickel column affinity chromatography and molecular exclusion chromatography.The sitting drop method in gas phase diffusion was used for crystal screening of Amuc＿1102 protein,and finally X-ray diffraction technology was used.The crystal structure of Amuc＿1102 protein was resolved,and the final resolution was 1.75 (?).The structure of Amuc＿1102 protein adopts Immunoglobulin(Ig)-like folding method,which is composed of two anti-parallel β-sheets,including nine β-strands and oneα-helix.Through structural comparison analysis,it was found that it has a similar folding pattern with the other three archaeal proteins(Pilin,FlaF,and FlaG)related to T4P-like structures,indicating that they may have similar functions.In addition,Amuc＿1102 exists as a trimer in both crystal structure and solution state,and the stability of the trimer structure in the crystal is also confirmed by point mutation experiments,which is different from the assembly method of Pilin,FlaF,and FlaG.This part of the research provides a structural basis for research and analysis to clarify the formation and assembly process of T4 P in A.muciniphila.Amuc＿1098 of the Amuc＿1098-Amuc＿1102 gene cluster encodes Pil Q in the T4 P assembly system as a channel protein.For the Amuc＿1098 protein,different sequences of truncation were designed and recombinant plasmids were constructed.Similarly,the Amuc＿1098 related protein was heterologously expressed and purified in E.coli,and the obtained protein was tried to grow crystals.The protein crystal of Amuc＿1098 protein has not yet been obtained.It is necessary to try the construction of different sequence fragments and the exploration of other experimental conditions to obtain protein crystals.After the structure is analyzed by diffraction,its function is further explained,and the T4 P mechanism of A.muciniphila can be better clarified through the structure.