A substrate translocation trajectory in a cytoplasm-facing topological model of the monocarboxylate/H+ symporter Jen1p

• Autor(a) principal: Silva, Isabel Soares
• Data de Publicação: 2011
• Outros Autores: Pessoa, Joana Sá, Myrianthopoulos, Vassilios, Mikros, Emmanuel, Casal, Margarida, Diallinas, George
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/1822/15406
• topic: Structure-function relationships; Lactate permease; Specificity; Molecular simulations; Substrate docking; Science & Technology

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H+ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence 379NXX[S/T]HX[S/T]QD387 in transmembrane segment VII (TMS-VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS-V and TMS-XI directly modify Jen1p function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are critical specificity determinants for the distinction of mono- from di-carboxylates, and N501 (TMS-XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to a protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS-II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking approaches reveal a ‘trajectory-like’ substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.