Role of flexibility and polarity as determinants of the hydration of internal cavities and pockets in proteins

Abstract

Molecular dynamics simulations of Staphylococcal nuclease and of 10 variants with internal polar or ionizable groups were performed to investigate systematically the molecular determinants of hydration of internal cavities and pockets in proteins. In contrast to apolar cavities in rigid carbon structures, such as nanotubes or buckeyballs, internal cavities in proteins that are large enough to house a few water molecules will most likely be dehydrated unless they contain a source of polarity. The water content in the protein interior can be modulated by the flexibility of protein elements that interact with water, which can impart positional disorder to water molecules, or bias the pattern of internal hydration that is stabilized. This might explain differences in the patterns of hydration observed in crystal structures obtained at cryogenic and room temperature conditions. The ability of molecular dynamics simulations to determine the most likely sites of water binding in internal pockets and cavities depends on its efficiency in sampling the hydration of internal sites and alternative protein and water conformations. This can be enhanced significantly by performing multiple molecular dynamics simulations as well as simulations started from different initial hydration states.

FIGURE 1

(Left) Crystal structure of the PHS/V66E variant showing the locations of the crystallographically resolved water molecules in the microcavity near position 66 and at two other sites, labeled A and B. (Right) Simultaneous representation of water-binding sites in the microcavity for all of simulated variants. Water molecules that are observed in crystal structures are shown in blue. Those that are only observed in DOWSER calculations are shown in red. Routes of water penetration and exit observed in MD simulations are indicated with arrows and labeled with Roman numerals I–VI.

FIGURE 2

Locations of internal water molecules observed in crystallographic structures (blue), or MD simulations (red) started from the crystal hydration states. Only water molecules that have residence times longer than 1 ns are shown. Only the results of a single MD simulation for each variant are represented in this figure.

FIGURE 3

Snapshots representing the three most common hydration patterns sampled during MD simulations of the V66E variant.

FIGURE 4

Region near Asn-66 in the crystal structure of PHS/V66N variant obtained at room temperature. 2F_o-F_c electron density map is in cyan, and 1F_o-F_c electron density map is in orange.