title: The respiratory syncytial virus transcription antiterminator M(2-1) is a highly stable, zinc binding tetramer with strong pH-dependent dissociation and a monomeric unfolding intermediate.
abstract: The human respiratory syncytial virus M(2-1) transcription antiterminator is an essential elongation factor required by the RNA polymerase for effective transcription beyond the first two nonstructural genes. Its exclusive presence in pneumovirus among all paramyxovirus suggests a unique function within this small genus. With the aim of understanding its biochemical properties, we investigated this α-helical tetramer by making use of a biophysical approach. We found that the tetramer hydrodynamic radius is considerably extended at high ionic strengths and determined its zinc content to be one atom per monomer. Dissociation-unfolding experiments show a fully reversible and concentration-dependent cooperative transition, but secondary and tertiary structural changes are uncoupled at lower protein concentrations. We detect the presence of a monomeric intermediate, which can be classified as a "late molten globule" with substantial secondary and tertiary structure. Global fittings of experiments from three different probes at two M(2-1) concentrations provide a free energy of dissociation-unfolding of -36.8 ± 0.1 kcal mol(-1), corresponding to a tight dissociation constant of 10(-28) M(3) at pH 7.0. The tetramer affinity is strongly governed by pH, with a free energy change of 13 kcal mol(-1) when pH decreases from 7.0 to 5.0 (K(D) = 10(-18) M(3)). The drastic changes that take place within a pH range compatible with a cellular environment strongly suggest a regulatory effect of pH on M(2-1) structure and biochemical properties, likely affecting transcription and interaction with proteins and RNA.
authors.affiliation: Protein Structure-Function and Engineering Laboratory, Fundación Instituto Leloir and IIBA-Conicet, Patricias Argentinas 435, 1405 Buenos Aires, Argentina.
authors.forename: Sebastián A
authors.initials: SA
authors.lastname: Esperante
authors.forename: Lucía B
authors.initials: LB
authors.lastname: Chemes
authors.forename: Ignacio E
authors.initials: IE
authors.lastname: Sánchez
authors.forename: Gonzalo
authors.initials: G
authors.lastname: de Prat-Gay
mesh_terms: D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D058767:Protein Unfolding; D018357:Respiratory Syncytial Virus Infections / Q000821:virology; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D010445:Peptide Elongation Factors; D014764:Viral Proteins; D015032:Zinc