Science for Health
01 May 2012
Scientists at NIMR have designed a mutant that eliminates nucleic acid binding by K-homology (KH) domains without destabilizing the KH fold, changing its structure or affecting KH–protein interactions. The research is published in Nucleic Acids Research.
KH-containing proteins are key components of gene regulation networks in eukaryotes. They play a key role in coordinating the different steps of RNA synthesis, metabolism and localisation. In the majority of the proteins of this class, RNA target recognition is mediated by multiple KH domains acting synergistically. A precise understanding of the role of the single KH domains is therefore necessary to dissect the regulatory networks at the molecular level. In functional studies mutagenesis is the best way to achieve this without perturbing the physiological structural environment, which often plays a role in recognition.
Scientists in the group led by Andres Ramos (pictured), in NIMR’s Division of Molecular Structure, in collaboration with Steve Martin in the Division of Physical Biochemistry, have designed a double mutation in the KH GxxG loop that eliminates nucleic acid binding without compromising the structure and stability of the domain. They have also provided a proof of principle for the use of the mutant, showing how mutation of KSRP links the sequence specificity of the different KH domains to their role in mRNA recognition and decay.
We have designed a robust molecular biology tool for the functional investigation of the roles of individual KH domains in nucleic acid binding proteins. It will report on RNA-binding by each KH domain in the context of the physiological structure and interactions. The tool should be useful for screening large numbers of protein–RNA interactions and to help rationalise the known ensembles of RNA targets of KH domains, de-convoluting the role of single domains in combinatorial recognition.
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The conserved GXXG loop of a KH domain contacts the charged nucleic acid backbone to orient the RNA nucleobases towards the protein’s hydrophobic groove. Although the precise details of binding vary among KH domains, as exemplified by the structures of RNA bound KH domains from Nova-2 (top) and SF1 (bottom), this interaction is universal and essential for nucleic acid binding.
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Introducing the GXXG to GDDG mutation into either of the KH domains within the ZBP1 KH3/KH4 di-domain results in the loss of RNA binding by the mutated domain. Shown here are overlaying fingerprint 15N-correlation NMR spectra recorded during the titrations of wild type and mutant forms of the protein with RNA. The side panels show two peaks originating from KH3 (V34) and KH4 (V119). Upon titrating RNA into the wild type protein both peaks move (middle) but when RNA is titrated into the KH3 or KH4 mutants (top and bottom) the peaks of the mutated domain are not affected.
KH domains with impaired nucleic acid binding as a tool for functional analysis
David Hollingworth, Adela M. Candel, Giuseppe Nicastro, Stephen R. Martin, Paola Briata, Roberto Gherzi and Andres Ramos (2012)
Nucleic Acids Research, Epub ahead of print. Fulltext.
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