Allosteric Signaling in the Biotin Repressor Occurs via Local Folding Coupled to Global Dampening of Protein Dynamics

Abstract

SummaryThe biotin repressor is an allosterically regulated site-specific DNA binding protein. Binding of the small ligand, bio-5'-AMP, activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand binding site concomitant with effector binding to the repressor monomer. In this work the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using HydrogenDeuterium exchange coupled to Mass Spectrometry. Measurements were performed on the apoprotein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from H-D exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with magnitude of the functional response to effector binding.

Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

“…The ability to retain biotinoyl-adenylate in the active site is also impaired (46), and the N-terminal deletion mutant is unable to grow at physiological biotin concentrations (unpublished data). Another example of interdomain communication is the restructuring of loops in the C-terminal domain observed upon binding of a biotinoyladenylate analogue to the catalytic domain (25,45). Among our mutants, the phenotypes of the I187T and K267M superrepressor BirAs probably reflect disturbances in interdomain communications.…”

The ensemble nature of allostery

“…The protein may have one or more domains or subunits whose structure can be determined, but which may show conformational dynamics when observed in NMR experiments. Many examples of structured allosteric proteins that show dynamic changes have been documented 4,18,23–26,72 . For reasons described below, these are ideal systems to probe ensemble properties.…”

Indirect DNA Readout on the Protein Side: Coupling between Histidine Protonation, Global Structural Cooperativity, Dynamics, and DNA Binding of the Human Papillomavirus Type 16 E2C Domain

“…This "indirect readout" can couple DNA binding to protein dimerization and other molecular functions through allosterism. 7,8,12,19,20 Human papillomaviruses (HPVs) are highly prevalent pathogens of epithelial tropism. 21,22 Mucosal HPV types are the etiological agents of cervical cancer, the second most common cancer in women, and of vaginal, anal, penile, and head and neck cancers.…”