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Importance of amino acids Leu135 and Tyr236 for the interaction between EhCFIm25 and RNA: a molecular dynamics
Juan David Ospina-Villa1, Juan García-Contreras1, Jorge Luis Rosas-Trigueros2
1Programa Institucional de Biomedicina Molecular, Programa de Doctorado en Ciencias en Biotecnología, ENMH, Instituto Politécnico Nacional, Guillermo Massieu Helguera 239, Fracc. La Escalera, Ticomán, Del. Gustavo A. Madero, CP 07320, Ciudad de México, Mexico.
Abstract:
The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.
Insights
The study reveals how Entamoeba histolytica CFIm25 (EhCFIm25) binds RNA, identifying key structural dynamics and lysine interactions with the GUUG sequence. This deepens understanding of polyadenylation in amoebiasis.
Area of Science:
- Molecular Biology
- Biochemistry
- Parasitology
Background:
- The CFIm25 subunit is crucial for mRNA 3' end polyadenylation, influencing factor recruitment and cleavage.
- Entamoeba histolytica, a protozoan parasite, causes human amoebiasis, and its homologous CFIm25 (EhCFIm25) plays a role in its biology.
- Previous work identified conserved residues Leu135 and Tyr236 in EhCFIm25 as important for RNA binding and the GUUG sequence as its recognition site.
Purpose of the Study:
- To elucidate the molecular interactions and network governing EhCFIm25's structure and function.
- To investigate the impact of mutations, particularly at Leu135, on EhCFIm25's RNA binding capabilities using molecular dynamics simulations.
- To provide a detailed structural and mechanistic understanding of EhCFIm25's RNA binding.
Main Methods:
- Molecular dynamics (MD) simulations were performed on wild-type (WT) EhCFIm25 and mutant proteins.
- Simulations included scenarios with proteins alone and interacting with the GUUG RNA sequence.
- Analysis focused on Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) to assess protein stability and dynamics, alongside contact analysis.
Main Results:
- WT EhCFIm25 converged to lower RMSD values more rapidly than mutants when bound to the GUUG sequence.
- RMSF analysis revealed similar amino acid movement patterns between WT and the EhCFIm25*L135T mutant, irrespective of GUUG binding.
- The EhCFIm25*L135T mutant, showing slight experimental RNA binding, exhibited comparable stabilization of bends and alpha helices to WT, with significant lysine contacts to the G4 nucleotide.
Conclusions:
- The study provides a clear description of the structural and mechanistic basis for EhCFIm25's RNA binding capacity.
- The Leu135 residue and specific lysine interactions are highlighted as critical for EhCFIm25 function.
- Findings contribute to understanding post-transcriptional regulation in Entamoeba histolytica and potential therapeutic targets.
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