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Technology Topics Crystallography

Drug Discovery: Identifying Dynamic Networks by CONTACT

SBKB [doi:10.1038/sbkb.2012.165]
Technical Highlight - October 2013
Short description: A new automated algorithm uses X-ray data to identify dynamic contact networks involved in enzyme catalysis.

Conformational exchange in cyclophilin A. Six contact networks comprising 29% of residues are mapped on the three-dimensional structure of CYPA. 1

Most enzymes fluctuate between different conformations in order to accomplish their function, and catalytic or regulatory mechanisms often involve long range motions. Although X-ray crystallography offers high-resolution views of proteins in alternate conformations, single or even multiple structures may not provide detailed information on the precise nature of allosteric communication between distant sites.

In an effort to harvest functional dynamics information from X-ray data, van den Bedem (PSI JCSG), Fraser and colleagues have now developed a new algorithm: contact networks through alternate conformation transitions (CONTACT). To identify interaction networks, qFit—an algorithm previously developed to extract conformational heterogeneity from X-ray diffraction data—is first used to define possible alternative conformations. CONTACT then uses this information to calculate the van der Waals interactions across all alternative conformations in order to define those likely to propagate to other residues. The identification of contact networks and analysis of their behavior when perturbed can provide insight into how conformational dynamics underlies enzyme catalysis.

As a validation test for CONTACT, the authors looked at two enzymes known to undergo significant conformational exchanges during catalysis: cyclophilin A (CYPA) and dihydrofolate reductase (DHFR). CONTACT identified multiple plausible transition pathways to known end states of CYPA, in line with recent NMR studies. To study long-range perturbation in DHFR, the authors obtained high-resolution X-ray diffraction datasets for wild-type DHFR at both cryogenic and room temperatures (PDB 4KJJ and 4KJK, respectively). They identified a contact network that connects the dynamic FG loop, the nicotinamide adenine dinucleotide phosphate (NADP)-binding pocket and the adenosine-binding domain of DHFR. CONTACT predicted that removal of NADP would disrupt coupling between the FG loop and adenosine-binding domain, a prediction experimentally confirmed by NMR analysis of DHFR containing the G121V mutation located within the FG loop. Similar analyses of another catalytically-defective DHFR mutant (PDB 3QL3 and 4KJL) unexpectedly revealed an expanded contact network resulting in non-productive motion around the active site and loss of catalytic efficiency.

This work demonstrates that X-ray data obtained at room temperature reveal accessible alternative conformations that are not observed at cryogenic temperatures. The development of methods allowing the collection of high-quality X-ray data at temperatures above the glass transition will therefore facilitate the predictions of allosteric pathways to their full extent. CONTACT should be a useful tool to complement the current arsenal of methods aimed at deciphering protein conformational dynamics.

Stéphane Larochelle


  1. H. van den Bedem et al. Automated identification of functional dynamic contact networks from X-ray crystallography.
    Nat Methods. 10, 896-902 (2013). doi:10.1038/nmeth.2592

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