The prokaryotic zinc-finger domain Ros87 (the DNA-binding region of the Ros protein from Agrobacterium tumefaciens) binds a structural Zn(II) ion and unfolds through a two-step process in which a metal-stabilized intermediate converts to the native state by a barrierless (downhill) transition, whereas the highly homologous but zinc-lacking domain Ml4_52-151 folds in a single fully cooperative two-state step. What remains open is the atomic-level mechanism of this metal-induced cooperativity switch: the atomic-resolution structure of the Zn(II)-bound intermediate ensemble, the rates and ligand-by-ligand coordination sequence of the Zn(II) association step that nucleates folding, the structural and thermodynamic determinants that select between reversible downhill conversion and irreversible metal loss at high temperature, and whether the switch is a general property of structural metalloproteins or specific to the Ros/MucR zinc-finger family.

Background

In the funneled energy-landscape picture of protein folding, a domain may fold in a single cooperative two-state transition (only native and unfolded ensembles populated at equilibrium) or through intermediates corresponding to additional local free-energy minima. Downhill folding denotes the limit in which one or more steps of the (un)folding process are barrierless, so that structure is lost or gained progressively over a temperature or denaturant range rather than in a sharp all-or-none transition; such behavior is considered rare because a substantial folding barrier normally protects proteins from aggregation and proteolysis. Metal-binding proteins constitute more than 30% of folded proteins, and in many of them — for example the zinc-finger class — the metal plays a purely structural role, raising the question of how cofactor binding reshapes the folding energy landscape.

Ros87 is an 87-residue domain (residues 56–142 of the Ros protein of Agrobacterium tumefaciens) that folds into a novel prokaryotic version of the Cys2His2 zinc finger: compared with the small eukaryotic zinc-finger modules, it forms a larger βββαα\beta\beta\beta\alpha\alpha globular domain with an extended hydrophobic core wrapped around the Zn(II) ion coordinated by two cysteines and two histidines (His37 and His42, separated by a four-residue spacer). Its NMR structure was reported in 2007. Highly homologous domains from Mesorhizobium loti, such as Ml1_53-149 (zinc-binding, different coordination sphere) and Ml4_52-151 (zinc-lacking), share the same overall fold.

Palmieri et al. (2013) combined far-UV circular dichroism (CD), differential scanning calorimetry (DSC), and NMR spectroscopy, including 1^1H/2^2H exchange (a residue-resolved probe of which amide protons are protected in structured regions), to compare the thermal unfolding of Ros87 with that of Ml4_52-151. They found that the zinc-lacking homolog unfolds fully cooperatively, whereas Zn(II)-bound Ros87 unfolds in two steps: a first transition populates a metal-binding intermediate, retaining native-like structure in the region comprising the β\beta-hairpin and part of the helices, and the intermediate then converts to the native state through a barrierless downhill transition — the first report of a structural metal ion inducing partly downhill folding, an observation unprecedented for any metal ion. The authors framed the finding as contributing to the search for cofactors' general roles in protein folding reactions, and left the detailed atomic structure of the intermediate, the kinetics of the Zn(II) association step, the determinants of the folding scenario, and the generality of the switch unsettled. At higher temperature Ros87 undergoes a further event attributed to loss of the metal ion, which is irreversible in phosphate buffer.

Problem Statement

Provide an atomic-level mechanistic account of how structural Zn(II) binding switches the folding of prokaryotic zinc-finger domains from fully cooperative two-state behavior (as in the zinc-lacking homolog Ml4_52-151) to a two-step process ending in a barrierless downhill conversion, jointly establishing: (1) the atomic-resolution structural ensemble of the Zn(II)-stabilized folding intermediate of Ros87, including the side-chain packing and which native ligands (among Cys24, Cys27, His37, His42) are coordinated in the intermediate; (2) the rates and microscopic coordination sequence of the Zn(II) association step that nucleates the intermediate, a step not yet directly resolved kinetically; (3) the structural and thermodynamic determinants — metal identity, coordination-sphere composition, and solvent conditions — that decide whether the metal-bound domain undergoes reversible barrierless intermediate-to-native conversion or irreversible metal loss at high temperature; and (4) whether this metal-induced cooperativity switch operates in structural metalloproteins beyond the Ros/MucR zinc-finger family. These four facets are jointly required, independently standing components of one objective — explaining, at the level of structures, energies, and rates, the mechanism by which a structural metal ion reshapes folding cooperativity — where facet (4) is the generality test of whether that mechanism is a property of structural metalloproteins beyond the Ros/MucR family rather than a family-specific peculiarity.

References

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