Crystal structure of the human O-6-alkylguanine-DNA alkyltransferase

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to O6-alkylation of guanine in DNA. This lesion results in transition mutations. In both prokaryotic and eukaryotic cells, repair is effected by direct reversal of the damage by a suicide protein, O6-alkylguanine-DNA alkyltransferase. The alkyltransferase removes the alkyl group to one of its own cysteine residues. However, this mechanism for preserving genomic integrity limits the effectiveness of certain alkylating anticancer agents. A high level of the alkyltransferase in many tumour cells renders them resistant to such drugs. Here we report the X-ray structure of the human alkyltransferase solved using the technique of multiple wavelength anomalous dispersion. This structure explains the markedly different specificities towards various O6-alkyl lesions and inhibitors when compared with the Escherichia coli protein (for which the structure has already been determined). It is also used to interpret the behaviour of certain mutant alkyltransferases to enhance biochemical understanding of the protein. Further examination of the various models proposed for DNA binding is also permitted. This structure may be useful for the design and refinement of drugs as chemoenhancers of alkylating agent chemotherapy

Combining X-ray and neutron crystallography with spectroscopy.

X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H(+) ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction

Combining X-ray and neutron crystallography with spectroscopy.

X-ray protein crystallography has, through the determination of the three-dimensional structures of enzymes and their complexes, been essential to the understanding of biological chemistry. However, as X-rays are scattered by electrons, the technique has difficulty locating the presence and position of H atoms (and cannot locate H(+) ions), knowledge of which is often crucially important for the understanding of enzyme mechanism. Furthermore, X-ray irradiation, through photoelectronic effects, will perturb the redox state in the crystal. By using single-crystal spectrophotometry, reactions taking place in the crystal can be monitored, either to trap intermediates or follow photoreduction during X-ray data collection. By using neutron crystallography, the positions of H atoms can be located, as it is the nuclei rather than the electrons that scatter neutrons, and the scattering length is not determined by the atomic number. Combining the two techniques allows much greater insight into both reaction mechanism and X-ray-induced photoreduction

Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency

Cytochrome P450BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase, is a member of a very large superfamily of monooxygenase enzymes. The available crystal structures of the enzyme show non-productive binding of substrates with their ω-end distant from the iron in a hydrophobic pocket at one side of the active site. We have constructed and characterised mutants in which this pocket is filled by large hydrophobic sidechains replacing alanine at position 82. The mutants having phenylalanine or tryptophan at this position have very much (~800-fold) greater affinity for substrate, with a greater conversion of the haem iron to the high-spin state, and similarly increased catalytic efficiency. The enzyme as isolated contains bound palmitate, reflecting this much higher affinity. We have determined the crystal structure of the haem domain of the Ala82Phe mutant with bound palmitate; this shows that the substrate is binding differently from the wild-type enzyme but still distant from the haem iron. Detailed analysis of the structure indicates that the tighter binding in the mutant reflects a shift in the conformational equilibrium of the substrate-free enzyme towards the conformation seen in the substrate complex rather than differences in the enzyme-substrate interactions. On this basis, we outline a sequence of events for the initial stages of the catalytic cycle. The Ala82Phe and Ala82Trp mutants are also very much more effective catalysts of indole hydroxylation than the wild-type enzyme, suggesting that they will be valuable starting points for the design of mutants to catalyse synthetically useful hydroxylation reactions

Example of multiple conformations of Q498 suggested in crystal structures.

The 2Fo-Fc electron density map (in blue, contoured at 1.5σ) and Fo-Fc difference map (contoured at -3σ (red) and green (+3σ)) are shown for the WT crystal structure of ACE2-RBD (PDB ID 6M0J [13]). The positive (green) density adjacent to the side chain of Q498 suggests this can exist in different conformations, thus weakening the interaction with neighbouring residues. Examination of the region of Q498 in PDB entries 7WQB, 7RPV, 7EFR, 7EFP, 7NXC, 7L0N, 7DMU and 6VW1 all show difference electron density adjacent to the side chain. PDB entries 7EKE, 7EKY, 7EKH, 7EKF and 6LZG all have the residue modelled in dual conformations. Only 7LO4 does not show disorder in Q498. (DOCX)</p

The Q498H mutation enables binding of SARS-CoV-2 RBD to rat ACE2.

(a) Kinetic binding constants for RBD binding to RaACE2 measured using biolayer interferometry. Binding was measured with RBD concentrations of 48, 90, 150, 300 and 600nM. Where no binding was detected an additional 1200nM RBD concentration was also tested for binding. RBD with the Q498H mutation binds rat receptor. Data are shown as means and SEM for at least three independent experiments. (b) Rat (RaACE2) and mouse ACE2 (MoACE2) differ from human ACE2 (HuACE2) in key residues contributing to SARS-CoV-2 RBD binding. Residues in human ACE2 at the RBD binding interface are aligned with the corresponding residues in rat and mouse ACE2. Residues that form hydrogen bonds or salt bridges with RBD are in bold and those that differ in rodent are in red. (c) Bonding interactions between Q24 and N487 (d) D30 and K417 (e) Y83 and N487 plus Y489, and (f) K353 and G496, in HuACE2 (yellow) and RBD (green).</p

Q498H and Q498R mutations modify binding of B.1.351 and B.1.617.1/3 variant RBD to HuACE2.

(a) Biolayer interferometry was performed with the indicated RBD in solution and HuACE2 immobilise on the sensor. Binding was measured with RBD concentrations of 12, 30, 60, 120nM, and an additional 6nM concentration in some cases. Curves were fitted and used to calculate Kon, Koff and KD. Data are shown as means and SEM for at least three independent experiments. (b) H498 and Y501 in SARS-CoV-2 RBD compete for interaction with Y41 in HuACE2 and the clash between residues is indicated by the overlapping Van der Waals surfaces. The proximity of the H498 side chain is shown with respect to the side chain of a Y501 inserted into our RBD:ACE2 complex structure. (c) R498 and Y501 side chain positions shown along with D38, Y41 and Q42 (PDB accession number 7BH9 [14]).</p

Effects of Gln498His and Gln498Arg mutations on binding of B.1.617.1/3 and B.1.351 variant RBD to RaACE2.

Effects of Gln498His and Gln498Arg mutations on binding of B.1.617.1/3 and B.1.351 variant RBD to RaACE2.</p

Cryo-EM data collection, refinement and validation statistics.

(DOC)</p

Structure of S477N/Q498H-RBD in complex with HuACE2.

Cryo-EM structure and model of the ACE2-RBD complex. Sharpened Cryo-EM map (a) of the HuACE2-S477N/Q498H-RBD complex with ACE2 coloured yellow and RBD in green. The refined coordinates shown in cartoon representation (b) and coloured as above. In panel (c) H498 can be seen in proximity to ACE2 Y41 forming a non-planar π-interaction while ACE2 residues K353 and D38 are within hydrogen bonding distance to H498 and could contribute to the tighter interaction formed by this RBD mutant, whereas Panel (d) shows Q498 in WH-RBD (PDB accession number 6M0J [13]) in proximity to ACE-2 Q42. Panel (e) indicates that the S477N mutation places this longer side-chain closer to S19 in ACE2 and within hydrogen bonding distance thus enhancing the binding between HuACE2 and S477N/Q498H-RBD. Panel (f) shows positioning of S477 in WH-RBD and ACE2 S19 (PDB accession number 6M0J [13]).</p