The main protease (3CL Mpro) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and ...His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys145 is in the negatively charged thiolate state and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment.
Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease ...(M
) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the M
/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor's keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the M
active site adapt to the inhibitor, which appears to be an intrinsic property of M
. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with M
which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.
Human manganese superoxide dismutase is a critical oxidoreductase found in the mitochondrial matrix. Concerted proton and electron transfers are used by the enzyme to rid the mitochondria of O
. The ...mechanisms of concerted transfer enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules at particular redox states. Here, neutron diffraction of two redox-controlled manganese superoxide dismutase crystals reveal the all-atom structures of Mn
and Mn
enzyme forms. The structures deliver direct data on protonation changes between oxidation states of the metal. Observations include glutamine deprotonation, the involvement of tyrosine and histidine with altered pK
s, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond. We report a concerted proton and electron transfer mechanism for human manganese superoxide dismutase from the direct visualization of active site protons in Mn
and Mn
redox states.
Potassium ion channels utilize a highly selective filter to rapidly transport K
ions across cellular membranes. This selectivity filter is composed of four binding sites which display almost equal ...electron density in crystal structures with high potassium ion concentrations. This electron density can be interpreted to reflect a superposition of alternating potassium ion and water occupied states or as adjacent potassium ions. Here, we use single wavelength anomalous dispersion (SAD) X-ray diffraction data collected near the potassium absorption edge to show experimentally that all ion binding sites within the selectivity filter are fully occupied by K
ions. These data support the hypothesis that potassium ion transport occurs by direct Coulomb knock-on, and provide an example of solving the phase problem by K-SAD.
The main protease (M
pro
) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound ...structures of M
pro
, a cysteine protease, have been determined, facilitating structure-based drug design. M
pro
plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, M
pro
contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV M
pro
, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 M
pro
as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of M
pro
are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N
δ
(HD) and N
ϵ
(HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 M
pro
in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.
The main protease (M
pro
) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics.
A cellulose synthesis complex with a "rosette" shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte ...algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.
Ion permeation in potassium ion channels Coates, Leighton
Acta crystallographica. Section D, Structural biology,
April 2020, 2020-Apr-01, 2020-04-01, 20200401, Letnik:
76, Številka:
4
Journal Article
Recenzirano
Odprti dostop
The study of ion channels dates back to the 1950s and the groundbreaking electrophysiology work of Hodgin and Huxley, who used giant squid axons to probe how action potentials in neurons were ...initiated and propagated. More recently, several experiments using different structural biology techniques and approaches have been conducted to try to understand how potassium ions permeate through the selectivity filter of potassium ion channels. Two mechanisms of permeation have been proposed, and each of the two mechanisms is supported by different experiments. The key structural biology experiments conducted so far to try to understand how ion permeation takes place in potassium ion channels are reviewed and discussed. Protein crystallography has made, and continues to make, key contributions in this field, often through the use of anomalous scattering. Other structural biology techniques used to study the contents of the selectivity filter include solid‐state nuclear magnetic resonance and two‐dimensional infrared spectroscopy, both of which make clever use of isotopic labeling techniques, while molecular‐dynamics simulations of ion flow through the selectivity filter have been enabled by the growing number of potassium ion channel structures deposited in the Protein Data Bank.
Key structural biology experiments that have sought to elucidate how potassium ions permeate and pass through the selectivity filter of potassium ion channels are reviewed.
SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (M
pro
) is essential for ...SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of M
pro
, a cysteine protease with a catalytic site comprising the noncanonical Cys145–His41 dyad, can help in guiding drug design. Here, a 2.0 Å resolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of M
pro
harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH
2
corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3′ by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free M
pro
demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for
in silico
modeling of the SARS-CoV-2 M
pro
catalytic mechanism.
The rise of neutron cryo‐crystallography Kwon, Hanna; Langan, Patricia S.; Coates, Leighton ...
Acta crystallographica. Section D, Structural biology,
August 2018, Letnik:
74, Številka:
8
Journal Article
Recenzirano
Odprti dostop
The use of boiled‐off liquid nitrogen to maintain protein crystals at 100 K during X‐ray data collection has become almost universal. Applying this to neutron protein crystallography offers the ...opportunity to significantly broaden the scope of biochemical problems that can be addressed, although care must be taken in assuming that direct extrapolation to room temperature is always valid. Here, the history to date of neutron protein cryo‐crystallography and the particular problems and solutions associated with the mounting and cryocooling of the larger crystals needed for neutron crystallography are reviewed. Finally, the outlook for further cryogenic neutron studies using existing and future neutron instrumentation is discussed.
The application of the cryogenic data‐collection environments used in protein X‐ray crystallography to neutron protein crystallography is discussed.
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