Structural basis of G-tract recognition by hnRNP F
Alternative splicing is a highly regulated mechanism generating proteomic diversity from a limited number of genes, and its disruption can cause numerous diseases. The heterogeneous nuclear ribonucleoprotein (hnRNP) F is involved in the regulation of mRNA metabolism by specifically recognizing G-tract RNA sequences. The mode of action of hnRNP F/H family members remains unclear and the molecular basis for G-tract recognition has not been determined. We have solved the solution structures of the three quasi RNA recognition motifs (qRRMs) of hnRNP F in complex with G-tract RNA. These structures show that qRRMs bind RNA in a very unusual manner, the G-tract being “encaged”, making the qRRM a novel RNA binding domain. The structures allowed us to define a consensus signature sequence for qRRMs and identify other human proteins containing qRRMs, which also recognize G-tracts. These structures explain how qRRMs can sequester G-tracts maintaining them in a single-stranded conformation. We also show that isolated qRRMs of hnRNP F are sufficient to regulate the alternative splicing of the apoptotic regulator Bcl-x. These results suggest that the encaging function of the qRRMs of hnRNP F is to prevent RNA structure formation that could impair splice site utilization.
Structural Model of the UbcH5B/CNOT4 complex
The protein CNOT4 possesses a N-terminal RING finger domain that acts as an E3 ubiquitin ligase and specifically interacts with UbcH5B, a E2 ubiquitin conjugating enzyme. We have identified the residues of UbcH5B important for the binding to CNOT4 RING domain by NMR chemical shift perturbation experiments and these data were used to generate structural models of the complex with the program HADDOCK. Together with the NMR data, additional biochemical data were included in a second docking and comparisons of the resulting model with the structure of the c-Cbl/UbcH7 complex revealed some significant differences, notably at specific residues, and gave structural insights into the E2/E3 specificity.
Dominguez C., Bonvin A.M.J.J., Winkler G.S., van Schaik F.M.A., Timmers H.Th.M. and Boelens R. (2004)
Structural model of the UbcH5B/CNOT4 complex revealed by combining NMR, mutagenesis and docking approaches
Structure, 12(4), 633-644
Abstract, Full text
HADDOCK: a protein- protein docking approach based on biochemical and/or biophysical information
The function of a protein often involves interactions with physiological partners (other protein, DNA, RNA, etc). There are several methods to study protein complexes (2 hybrid screening, fluorescence studies, etc) but few of these techniques give information at a structural level. X-ray and NMR, which are the best protein structure calculation methods, encounter difficulties solving complex structures, especially for transient and flexible complexes. Indeed, by X-Ray, the dynamic of the complex limits the formation of crystals and the size limitation of NMR is also a major limitation when considering high molecular weight complexes. New methods to study protein complexes at a structural level, called protein docking, are now emerging and have been well developed during the past few years. There are now a number of programs performing protein-protein docking.
By NMR, performing a chemical shift perturbation experiment of a complex formation is simple and very useful. The amino acids responsible for the complex formation can easily be identified but no data about the orientation of one protein relative to the other one is available.
We have therefore developed a new docking program, HADDOCK (High Ambiguity Driven protein –protein DOCKing) that directly uses experimental information to drive the docking process. We developed an AIR (Ambiguous Interaction Restraint) based on NMR titration data to simplify the docking calculation. The calculation is done in three steps:
- A rigid body docking
- A simulated annealing in torsion angle space protocol
- A refinement in explicit water
At each step, the structures are ranked in term of the interface energy (electrostatic, Van der Waals and ACS) and the best ones are used for the next step.
We have demonstrated the accuracy of our program with 4 molecular complexes where the structure of the free form of each protein and the structure of the complexes were known. Furthermore, for three of these complexes, the NMR titration data and for the fourth one, mutagenesis data were available. In all the case, the best structures generated by HADDOCK –i.e. the structures with the lowest interaction energy term- were the closest in term of RMSD (between 2 and 4 Å) to the published structure of the complex.
Dominguez C., Boelens R. and Bonvin A.M.J.J. (2003)
HADDOCK: A Protein-Protein Docking Approach Based on Biochemical or Biophysical Information
J. Am. Chem. Soc., 125(7) pp. 1731 - 1737
Abstract, Full text