The analysis of the SAXS data has provided us with the ab initio models of both the trimeric and the pentameric complexes, displayed in . Without the tRNAs the protein components of the Arc1p-complex appear to point in different directions and the proteins themselves look stretched with none or only very few interactions between their individual domains. There is still some empty volume within the overall shapes left after fitting the structure homologues inside the surface of the trimeric complex. This can be explained by the fact that we did not find structural homologues with the same number of amino acids as our S. cerevisiae proteins. All the individual components have a number of residues missing Arc1p 82 (residues 122–204), GluRS 36 (residues 181–205 and 711–723) and MetRS 47 (residues 705–751). Potential flexibility of the extremely extended trimeric complex may also play a role, but, overall, the extended shapes of the proteins can be well matched by the available high-resolution portions. The fidelity of the obtained model of the ternary complex is especially high given that it fits simultaneously six independently measured SAXS patterns.
The binding of the two tRNAs causes a significant compaction, literally a collapse, of the pentameric complex compared to the trimeric. The complex with the tRNAs is very compact, and even its apparent (Porod) volume is smaller than the volume without the tRNAs. This is explained by two reasons. First, the pentameric complex is a heterogeneous molecule (consisting of two components with significantly different X-ray contrast, proteins and RNA).These density fluctuations lead to additional scattering at higher angles and thus a smaller effective volume. Second and perhaps most important, the volume as seen by SAXS corresponds to the volume of the unbound solvent excluded by the molecule. The very extended trimeric complex is able to exclude more solvent (because of potential flexibility, higher hydration and electrostatic interactions) than the rather compact and rigid pentameric complex. This considerable volume compaction indicates substantial conformational changes of all three proteins when the tRNAs are bound. The arrangement of the individual domains inside the pentameric shape is not as straightforward as it is for the trimeric complex. It is not possible to assign a particular part of the shape to a specific protein, because of the dramatic volume compaction of the pentameric complex. However, by using the results of previous studies (6
) and the surface charges of the specific proteins as additional constraints, we can derive a sensible model for the structural assembly within the pentamer. The structures of the homologues fit nearly perfectly inside the pentameric envelope resulting from SAXS experiments. The fact, that there are some minor parts of the structures outside the ab initio
shape (shown in grey), may be a consequence of the relatively low sequence identities between the S. cerevisiae
proteins and the structural homologues, ranging from 23% to 45.3%.
The compaction observed upon addition of tRNA indicates the protein components of the ternary complex undergo substantial conformational changes upon the formation of the pentameric complex. The most plausible cause of these changes are electrostatic interactions between the proteins and the tRNA. Indeed, the analysis of the charge distribution of the synthetases and of Arc1p within their TRBDs show positive-charged surfaces, where the tRNAs can bind (). These interactions, given the extended shapes of the free proteins, may lead to significant compaction upon the binding of the synthetases to their cognate tRNA.
In the present work we report a totally unexpected effect, namely a drastic compaction of a protein complex upon tRNA binding. A compaction effect of such magnitude, literally a collapse, has, to our knowledge, not been reported before for nucleoprotein complexes. These impressive rearrangements of the subunits are most probably due to the surface charges of the individual protein domains. Electrostatic repulsion of the positively charged tRNA-binding domains of the synthetases as well as Arc1p may cause the star-like arrangement of the proteins in the trimeric complex. In the pentamer, on the other hand, the negatively charged tRNA backbones will induce a mutual contraction of these domains. The flexible links connecting the domains facilitate these rearrangements.
In yeast as well as in eukaryotic cells, it is important to have an effective translation machinery connected to a high degree of complex formation, because of the size and complexity of the cell. The affinity of binding the tRNAs is increased in the pentameric complex compared to the synthetase–tRNA complex alone, which shows the importance of the pentameric complex for the yeast cell. The aminoacylation of the tRNA is faster and more effective and therewith the whole translation machinery can be faster. Also the specificity to the tRNA is higher, because of the additional binding sites to the cofactor Arc1p. The observed structural results have therefore clear functional implications in the context of the efficiency of the translational mechanism.