Structural polymorphism of a marginally stable 4‐α‐helical bundle. Images of a trapped molten globule?

The general experience from structural studies of single amino-acid-substituted mutant proteins is that the effect of mutation is rather localized and minor. We have reported an exception to this rule by showing that a single alanine to proline substitution at position 31 of the Rop protein is sufficient for changing the topology of this 4-helical bundle (Fig. 1), leading to drastic changes both in its surface properties and the packing of its hydrophobic core. The original structure determination of this mutant was achieved through the analysis of an orthorhombic crystal form. A few puzzling observations were made at that time concerning both the mutant structure itself and some of its functional properties. The first observation concerned the reduced number (and density) of hydrophobic core packing interactions. This was manifested and reinforced by (i) the destruction of the layered structure of the hydrophobic core as seen in the wild-type protein; and (ii) by the absence of interactions between hydrophobic residues obeying a certain sequence periodicity (known as heptad repeat) which characterizes associating -helices. A related second observation concerned the large number of buried hydrophobic residues (approximately 30% of the total number of buried residues) for which the electron density maps suggested the presence of static or discrete disorder. The third was the presence of a large continuous cavity buried in the middle of the protein (and effectively separating the hydrophobic core in two discrete patches) with a volume of approximately 270 Å. Lastly, the finding that this mutant retained some of the biological activity of the wild-type protein could not be reconciled with the large movements (of the order of approximately 4 Å) that would be required to bring some side-chains (like Phe14, known to be essential for RNA-binding) into a suitable (for RNA-binding) distance. In the hope that we could address some of these questions, we pursued our attempts to determine the structure of a second (monoclinic) crystal form of A31P. It was only very recently that we succeeded in completing the crystallographic analysis of this second crystal form (and this only after we resorted to a computationally expensive 23-dimensional molecular replacement search). As shown in Figure 1, the monoclinic A31P crystal structure retained the same topology and folding motif as its orthorhombic counterpart, but everything else appears to be different: The relative juxtapositions of the helices deviate so much between the two structures, that when any one of them is used for superposition, none of the remaining three helices overlap. When the whole bundles are superimposed, the root mean square deviation (RMSD) between equivalent C atoms is approximately 3 Å. Not surprisingly, these differences are accompanied by changes in the mode of hydrophobic core packing and the bundles’ surface properties (data not shown). This in spite of the fact that the only consistent difference between the crystallization conditions of the two crystal forms is an increase of the pH by half a unit for the monoclinic form. The large differences between the two crystal forms (indicative of conformational polymorphism) together with (i) the presence of a mobile, loosely packed hydrophobic core; (ii) the presence of a large continuous (and probably solvent-exposed) hydrophobic cavity in the middle of the protein’s interior; and (iii) the greatly reduced stability of this mutant (compared with the wild-type protein), led us to hypothesize that A31P at its equilibrium state may have molten-globule-like characteristics. Clearly, such a proposition can not be supported solely by evidence obtained through the analysis of two static images, especially when these images have been determined from crystal structures with completely different packing environments. For this reason, and in an attempt to obtain evidence concerning the behavior of this mutant in solution and to further characterize its equilibrium properties, a 3-ns molecular dynamics simulation of A31P in explicit water was performed as described in the legend to Figure 2. Furthermore, and in order to have a measure of comparison for the analyses of the molecular dynamics results, similar 3-ns simulations were performed on wild-type Rop