Accessibility to internal cavities and ligand binding sites monitored by protein crystallographic thermal factors

Abstract

Protein structures are flexible both in solution and in the solid state. X-ray crystallographically determined thermal factors monitor the flexibility of protein atoms. A method utilizing such factors is proposed to delineate protein regions through which a ligand can exchange between binding site and bulk solvent. It is based on the assumption that thermally excited protein regions are excellent candidates for opening a ligand channel. Computationally simple and inexpensive, the method analyzes directions from which thermal factors can propagate within the protein, resulting in thermal motion paths (TMPs). Applications to engineered T4 lysozymes, where an artificial internal cavity can host hydrophobic molecules, and to sperm whale myoglobins, where the active site is completely buried, yielded results in agreement with other independent structural observations and with previous hypotheses. Further new features could also be suggested. The proposed TMP analysis could aid molecular dynamics simulation studies as well as time-resolved and site-directed mutagenesis experimental studies, especially given its modest computational expense and its direct roots in experimental results based on thermal factors determined in high-resolution crystallographic studies.

Pathways and Mechanisms for Product Release in the Engineered Haloalkane Dehalogenases Explored Using Classical and Random Acceleration Molecular Dynamics Simulations

“…3,4,8,10,17,18,20,34,41,46,72,73 The exchange through the transient tunnels corresponds to exchange through a so-called naturally fluctuating bottleneck, 74 which is a common mechanism to transiently enable access and egress of ligands in and out of the active site in the regions of lower density of protein atoms. This mechanism was previously reported for cytochrome P450, 42,44,47,59 acetylcholinesterase, 66,71 NADH oxidase, 19 T4 lysosyme, 75 and horseradish peroxidase. 69 Passage of the ligands through the protein matrix is a welldocumented phenomenon for gas migration in heme proteins.…”

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

“…To probe the conformational changes for substrate access to and exit from the active site more explicitly, we performed two types of analysis: thermal pathway analysis and molecular dynamics simulation (54). In thermal pathway analysis, the measured temperature factors in the crystal structures are analyzed to identify flexible regions where ligand channels may open (55). This analysis for cytochrome P450 cam indicates three particularly mobile regions of the protein as candidates for ligand channels.…”

Functional relevance of the internal hydrophobic cavity of urate oxidase

“…It is very hydrophobic, lined with almost 90% of carbon atoms. It is well known that internal cavities within proteins are crucial for conformational flexibility and domain motion [10,11] and accessibility for small ligands can only be granted through thermal motion [12]. Flexibility is thought to be necessary for functional efficiency.…”