The large groove in Tim50IMS
contains several exposed negatively charged residues (). We speculate that this groove is ideally suited as binding site for positively charged presequences/preproteins (additionally, hydrophobic residues located around the rim of the groove may contribute to an interaction with the hydrophobic surfaces of the amphipathic presequences), though an involvement of the groove in the cooperation of Tim50 with the TOM complex or further TIM subunits cannot be excluded. The crystal structure of Tim50IMS
was determined by molecular replacement using the phosphatase Scp1 that is specific for the C-terminal domain of RNA polymerase II.24
Scp1 is the top structural homologue of Tim50IMS
(Z-score of 22.8), exhibiting similar protein folding with rms derivation of 2.2 Å for the main chain atoms () (the sequence identity between human Scp1 and yeast Tim50IMS
is 33.5% [calculated by ClustalW]). Interestingly, the crystal structure of Scp1 was solved in complex with the C-terminal phosphorylated peptide of RNA Polymerase II.24
The peptide forms a U-turn in a groove that corresponds to the putative presequence-binding groove in Tim50IMS
(). In Tim50IMS
, the protruding β-hairpin swings away from the groove, providing more space for the presequence-binding groove to likely accommodate an α-helix formed by the presequence. The bottom of the peptide-binding groove of Scp1 contains a DXDX(T/V) phosphatase motif24
that is not present in Tim50 (and Tim50 does not show phosphatase activity).15
The bottom of the putative presequence-binding groove of Tim50IMS
is only weakly conserved (), which may reflect that Tim50 has to interact with a large variety of mitochondrial presequences.
Superimposition of Tim50IMS (green) and Scp1 structures (blue). The orientation of Tim50 is similar to that in . The bound peptide substrate of Scp1 is shown in red.
The close proximity between the putative preprotein-binding groove and the Tim23-interacting β-hairpin of Tim50 can provide a molecular explanation for the observation that binding of preproteins to Tim50 depends on the interaction of Tim50 with Tim23.21
Though NMR spectroscopy analysis indicated that Tim23IMS
is intrinsically disordered,25
recent studies provided evidence that the Tim23 sites for preprotein binding and interaction with Tim50 are in close proximity. Crosslinking studies mapped a region of Tim23 including residues 70 and 71 interacting with Tim50.20,22
NMR spectroscopy suggested that a Tim23 region from residues 71–84 is involved in presequence binding.25
We propose that for Tim50 as well as Tim23, the site of preprotein recognition is in close proximity to the Tim50-Tim23 interaction site, leading to a working model that the receptor module of the presequence translocase is formed by a composite presequence binding pocket that involves both Tim50 and Tim23.
In summary, our structural and functional analysis of Tim50 revealed that the protruding β-hairpin is important for recruiting Tim23, supporting the hypothesis that Tim50 and Tim23 function cooperatively to direct preproteins to the transmembrane channel formed by the C-terminal domain of Tim23.