Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

Determinants for the recognition of a mitochondrial presequence by the mito chondrial processing peptidase (MPP) have been investigated using mutagenes is and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at positi on -2 or -3, and the third class does not have any conserved arginines. Seq uence logos revealed loosely conserved cleavage motifs for the first two cl asses but no significant amino acid conservation for the third class. Inves tigation of processing determinants for a class III precursor, Nicotiana pl umbaginifolia F(1)beta precursor of ATP synthase (pF(1)beta), was performed using a series of pF(1)beta presequence mutants and mutant presequence pep tides derived from the C-terminal portion of the presequence. Replacement o f -2 Gln by Arg inhibited processing, whereas replacement of either the mos t proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory eff ect. The C-terminal portion of the pF(1)beta presequence forms a helix-turn -helix structure. Mutations disturbing or prolonging the helical element up stream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF(1)beta presequence peptide were built by homology modelling and empirical conformational energy search meth ods, respectively. Molecular docking of the pF(1)beta presequence peptide t o the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha -MPP and beta -MPP subunits in close prox imity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta - MPP. Our results show for the first time that the amino acid at the -2 posi tion, even if not an arginine, as well as structural properties of the C-te rminal portion of the presequence are important determinants for the proces sing of a class III precursor by MPP.