Alanine scanning of MS2 coat protein reveals protein-phosphate contacts involved in thermodynamic hot spots.
The co-crystal structure of the MS2 coat protein dimer with its RNA operator reveals eight amino acid side-chains contacting seven of the RNA phosphates. These eight amino acids and five nearby control positions were individually changed to an alanine residue and the binding affinities of the mutant proteins to the RNA were determined. In general, the data agreed well with the crystal structure and previous RNA modification data. Interestingly, amino acid residues that are energetically most important for complex formation cluster in the middle of the RNA binding interface, forming thermodynamic hot spots, and are surrounded by energetically less relevant amino acids. In order to evaluate whether or not a given alanine mutation causes a global change in the RNA-protein interface, the affinities of the mutant proteins to RNAs containing one of 14 backbone modifications spanning the entire interface were determined. In three of six protein mutations tested, thermodynamic coupling between the site of the mutation and RNA groups that can be even more than 16 A away was detected. This suggests that, in some cases, the mutation may subtly alter the entire protein-RNA interface.
Hobson
Dagmar
D
Department of Biochemistry, Molecular Biology, Cell Biology, Northwestern University 2205 Tech Drive, Hogan 2-100, Evanston, IL 60208, USA.
Uhlenbeck
Olke C
OC
D000409:Alanine / Q000235:genetics*; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D017909:Levivirus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D036022:Capsid Proteins; D010710:Phosphates; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D000409:Alanine