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.