Ultrahigh resolution drug design I: Details of interactions in human aldose reductase–inhibitor complex at 0.66 Å

Abstract

The first subatomic resolution structure of a 36 kDa protein [aldose reductase (AR)] is presented. AR was cocrystallized at pH 5.0 with its cofactor NADP+ and inhibitor IDD 594, a therapeutic candidate for the treatment of diabetic complications. X-ray diffraction data were collected up to 0.62 A resolution and treated up to 0.66 A resolution. Anisotropic refinement followed by a blocked matrix inversion produced low standard deviations (<0.005 A). The model was very well ordered overall (CA atoms' mean B factor is 5.5 A2). The model and the electron-density maps revealed fine features, such as H-atoms, bond densities, and significant deviations from standard stereochemistry. Other features, such as networks of hydrogen bonds (H bonds), a large number of multiple conformations, and solvent structure were also better defined. Most of the atoms in the active site region were extremely well ordered (mean B approximately 3 A2), leading to the identification of the protonation states of the residues involved in catalysis. The electrostatic interactions of the inhibitor's charged carboxylate head with the catalytic residues and the charged coenzyme NADP+ explained the inhibitor's noncompetitive character. Furthermore, a short contact involving the IDD 594 bromine atom explained the selectivity profile of the inhibitor, important feature to avoid toxic effects. The presented structure and the details revealed are instrumental for better understanding of the inhibition mechanism of AR by IDD 594, and hence, for the rational drug design of future inhibitors. This work demonstrates the capabilities of subatomic resolution experiments and stimulates further developments of methods allowing the use of the full potential of these experiments.

On the use of logarithmic scales for analysis of diffraction data

“…This confirms that the value of 1.7 Å initially reported at a home source (Lamour et al, 1999) was below what might be obtained. At the same time, (10) does not predict that some aldose reductase crystals can diffract to 0.66 Å resolution (Howard et al, 2004). Nevertheless, the possibility of similarly high-resolution data can be predicted for other crystals.…”

The active site of hen egg-white lysozyme: flexibility and chemical bonding

“…Refinements of multipole parameters are only possible if accurate X-ray diffraction data have been measured at low temperature (T < 100 K) and to high resolution (d min , preferably below 0.50 Å ). A single protein (crambin; Jelsch et al, 2000;Schmidt et al, 2011) diffracts to this limit, while subatomic resolution data sets of several more proteins have been measured (Ko et al, 2003;Howard et al, 2004;Wang et al, 2007). Here, we present an analysis of the d min = 0.65 Å data set of HEWL as published by Wang et al (2007).…”

High-resolution crystallography and drug design

“…all carbon atoms must have a total valence 4) or to characterize the nature of a given bond. We have already established the constancy of the total valence in peptide bonds in aldose reductase by the highly correlated behavior of the C --O and C-N bond distances (Howard et al, 2004). This analysis reveals the true nature of the peptide bond which varies from near-double bond to single bond depending on the micro-environment.…”

“…The study of these structures has also greatly improved and algorithms for multipolar and quantum modeling analysis have been devised. Furthermore, since 1998, several structures have been solved with a resolution better than 0.80 Å (ultra-high-resolution), in particular crambin (Jelsch et al, 2000), subtilisin (Kuhn et al, 1998) and aldose reductase (Howard et al, 2004). At this resolution the hydrogen atoms and the bond densities are clearly visible, and the atomic errors of co-ordinates are reduced even further ( $ 0.003 Å ), which makes the observed stereochemical differences highly significant.…”