Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies.

The recently reported "UK variant" (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments.

Ultrafast Magic-Angle Spinning: Benefits for the Acquisition of Ultrawide-Line NMR Spectra of Heavy Spin-1/2 Nuclei.

The benefits of the ultrafast magic-angle spinning (MAS) approach for the acquisition of ultrawide-line NMR spectra-spectral simplification, increased mass sensitivity allowing the fast study of small amounts of material, efficient excitation, and application to multiple heavy nuclei-are demonstrated for tin(II) oxide (SnO) and the tin complex [(LB)Sn(II) Cl](+) [Sn(II) Cl3 ](-) [LB=2,6-diacetylpyridinebis(2,6-diisopropylanil)] containing two distinct tin environments. The ultrafast MAS experiments provide optimal conditions for the extraction of the chemical-shift anisotropy tensor parameters, anisotropy, and asymmetry for heavy spin-1/2 nuclei.

The common structural architecture of Shigella flexneri and Salmonella typhimurium type three secretion needles.

The Type Three Secretion System (T3SS), or injectisome, is a macromolecular infection machinery present in many pathogenic Gram-negative bacteria. It consists of a basal body, anchored in both bacterial membranes, and a hollow needle through which effector proteins are delivered into the target host cell. Two different architectures of the T3SS needle have been previously proposed. First, an atomic model of the Salmonella typhimurium needle was generated from solid-state NMR data. The needle subunit protein, PrgI, comprises a rigid-extended N-terminal segment and a helix-loop-helix motif with the N-terminus located on the outside face of the needle. Second, a model of the Shigella flexneri needle was generated from a high-resolution 7.7-Å cryo-electron microscopy density map. The subunit protein, MxiH, contains an N-terminal α-helix, a loop, another α-helix, a 14-residue-long ß-hairpin (Q51-Q64) and a C-terminal α-helix, with the N-terminus facing inward to the lumen of the needle. In the current study, we carried out solid-state NMR measurements of wild-type Shigella flexneri needles polymerized in vitro and identified the following secondary structure elements for MxiH: a rigid-extended N-terminal segment (S2-T11), an α-helix (L12-A38), a loop (E39-P44) and a C-terminal α-helix (Q45-R83). Using immunogold labeling in vitro and in vivo on functional needles, we located the N-terminus of MxiH subunits on the exterior of the assembly, consistent with evolutionary sequence conservation patterns and mutagenesis data. We generated a homology model of Shigella flexneri needles compatible with both experimental data: the MxiH solid-state NMR chemical shifts and the state-of-the-art cryoEM density map. These results corroborate the solid-state NMR structure previously solved for Salmonella typhimurium PrgI needles and establish that Shigella flexneri and Salmonella typhimurium subunit proteins adopt a conserved structure and orientation in their assembled state. Our study reveals a common structural architecture of T3SS needles, essential to understand T3SS-mediated infection and develop treatments.

Studies on the MxiH protein in T3SS needles using DNP-enhanced ssNMR spectroscopy.

Bacterial T3SS needles formed by the protein MxiH are studied using DNP-enhanced ssNMR spectroscopy at 14.1 T (600 MHz). This technique provides spectra of good resolution, allowing us to draw conclusions about the protein dynamics. With the obtained signal enhancement, samples of limited quantity now get within reach of ssNMR studies.

Lewis base mediated autoionization of GeCl2 and SnCl2.

Cationic and anionic species of heavier low-valent group 14 elements are intriguing targets in main group chemistry due to their synthetic potential and industrial applications. In the present study, we describe the synthesis of cationic (MCl(+)) and anionic (MCl(3)(-)) species of heavier low-valent group 14 elements of germanium(II) and tin(II) by using the substituted Schiff base 2,6-diacetylpyridinebis(2,6-diisopropylanil) as Lewis base (LB). Treatment of LB with 2 equiv of GeCl(2)·dioxane and SnCl(2) in toluene gives compounds [(LB)Ge(II)Cl](+)[Ge(II)Cl(3)](-) (1) and [(LB)Sn(II)Cl](+)[Sn(II)Cl(3)](-) (2), respectively, which possess each a low-valent cation and an anion. Compounds 1 and 2 are well characterized with various spectroscopic methods and single crystal X-ray structural analysis.

Structural and biochemical rationale for enhanced spike protein fitness in delta and kappa SARS-CoV-2 variants.

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a structural head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation and with unknown biological implications. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

Preparation of RSn(I)-Sn(I)R with two unsymmetrically coordinated Sn(I) atoms and subsequent gentle activation of P4.

This article reports the reduction of [{2,6-iPr(2)C(6)H(3)NC(CH(3))}(2)C(6)H(3)SnCl] (1) with potassium graphite to afford a new distannyne [{2,6-iPr(2)C(6)H(3)NC(CH(3))}(2)C(6)H(3)Sn](2) (2) with a Sn-Sn bond. The most striking phenomenon of 2 is the presence of two differently coordinated Sn atoms (one is three-coordinated, the other is four-coordinated). The Sn-Sn bond length in 2 is 2.8981(9) Å, which is very close to that of a Sn-Sn single bond (2.97-3.06 Å). To elucidate the nature of the Sn-Sn bond, DFT calculation is carried out that shows there is no multiple bond character in 2. Furthermore, the reaction of 2 with white P(4) affords the tetraphosphabicylobutane derivative 3. This is the first example of gentle activation of white phosphorus by a compound with low valent Sn atoms. Note that, unlike 2, in 3 both Sn atoms are four-coordinated.

Progress in correlation spectroscopy at ultra-fast magic-angle spinning: basic building blocks and complex experiments for the study of protein structure and dynamics.

Recent progress in multi-dimensional solid-state NMR correlation spectroscopy at high static magnetic fields and ultra-fast magic-angle spinning is discussed. A focus of the review is on applications to protein resonance assignment and structure determination as well as on the characterization of protein dynamics in the solid state. First, the consequences of ultra-fast spinning on sensitivity and sample heating are considered. Recoupling and decoupling techniques at ultra-fast MAS are then presented, as well as more complex experiments assembled from these basic building blocks. Furthermore, we discuss new avenues in biomolecular solid-state NMR spectroscopy that become feasible in the ultra-fast spinning regime, such as sensitivity enhancement based on paramagnetic doping, and the prospect of direct proton detection.

Binding mechanism of an SH3 domain studied by NMR and ITC.

Complexes between Src-homology 3 domains and proline-rich target peptides can have lifetimes on the order of milliseconds, making them too short-lived for kinetic characterization by conventional methods. Nuclear magnetic resonance (NMR) dynamics experiments are ideally suited to study such rapid binding equilibria, and additionally provide information on partly bound intermediate states. We used NMR together with isothermal titration calorimetry (ITC) to characterize the interaction of the SH3 domain from the Fyn tyrosine kinase with a 12-residue peptide at temperatures between 10 and 50 degrees C. NMR data at all temperatures are consistent with an effectively two-state binding reaction, such that any intermediates are either very weakly populated or exchange extremely rapidly with the free or bound forms. Dissociation rate constants, determined by CPMG and ZZ-exchange NMR experiments, range from k(off)(10 degrees C) = 4.5 s(-1) to k(off)(50 degrees C) = 331 s(-1). ITC data at all temperatures follow a simple two-state interaction model. Binding is favored enthalpically, with a dissociation enthalpy, DeltaH(D)(30 degrees C) = 15.4 kcal mol(-1), and disfavored entropically, with a dissociation entropy, DeltaS(D)(30 degrees C) = 20.0 cal mol(-1) K(-1). The free protein and peptide have significantly higher heat capacity than the bound complex, DeltaC(p) = 352 cal mol(-1) K(-1), which is consistent with the largely hydrophobic character of the binding interface. An Eyring plot of k(off) values gives an activation enthalpy of dissociation, DeltaH(D)(double dagger)(30 degrees C) = 19.3 kcal mol(-1) and exhibits slight curvature consistent with the ITC-derived value of DeltaC(p). The curvature suggests that nonpolar residues of the hydrophobic interface are solvated in the transition state for dissociation. Association rate constants were calculated as k(on) = k(off)/K(D), and range from k(on)(10 degrees C) = 1.03 x 10(8) M(-1) s(-1) to k(on)(50 degrees C) = 2.0 x 10(8) M(-1) s(-1), with an apparent activation enthalpy, DeltaH(A)(double dagger) = 3.4 kcal mol(-1). Both the magnitudes and temperature dependence of k(on) values are consistent with a diffusion-limited association mechanism. The combination of NMR and ITC data sheds light on how the Fyn tyrosine kinase is activated by binding to proline-rich targets, and represents a powerful approach for characterizing transient protein/ligand interactions.

Structure determination of supra-molecular assemblies by solid-state NMR: Practical considerations.

In the cellular environment, biomolecules assemble in large complexes which can act as molecular machines. Determining the structure of intact assemblies can reveal conformations and inter-molecular interactions that are only present in the context of the full assembly. Solid-state NMR (ssNMR) spectroscopy is a technique suitable for the study of samples with high molecular weight that allows the atomic structure determination of such large protein assemblies under nearly physiological conditions. This review provides a practical guide for the first steps of studying biological supra-molecular assemblies using ssNMR. The production of isotope-labeled samples is achievable via several means, which include recombinant expression, cell-free protein synthesis, extraction of assemblies directly from cells, or even the study of assemblies in whole cells in situ. Specialized isotope labeling schemes greatly facilitate the assignment of chemical shifts and the collection of structural data. Advanced strategies such as mixed, diluted, or segmental subunit labeling offer the possibility to study inter-molecular interfaces. Detailed and practical considerations are presented with respect to first setting up magic-angle spinning (MAS) ssNMR experiments, including the selection of the ssNMR rotor, different methods to best transfer the sample and prepare the rotor, as well as common and robust procedures for the calibration of the instrument. Diagnostic spectra to evaluate the resolution and sensitivity of the sample are presented. Possible improvements that can reduce sample heterogeneity and improve the quality of ssNMR spectra are reviewed.

Recovery of bulk proton magnetization and sensitivity enhancement in ultrafast magic-angle spinning solid-state NMR.

The sensitivity of solid-state NMR experiments is limited by the proton magnetization recovery delay and by the duty cycle of the instrument. Ultrafast magic-angle spinning (MAS) can improve the duty cycle by employing experiments with low-power radio frequency (RF) irradiation which reduce RF heating. On the other hand, schemes to reduce the magnetization recovery delay have been proposed for low MAS rates, but the enhancements rely on selective transfers where the bulk of the (1)H magnetization pool does not contribute to the transfer. We demonstrate here that significant sensitivity enhancements for selective and broadband experiments are obtained at ultrafast MAS by preservation and recovery of bulk (1)H magnetization. We used [(13)C, (15)N]-labeled glutamine as a model compound, spinning in a 1.3 mm rotor at a MAS frequency of 65 kHz. Using low-power (1)H RF (13.4 kHz), we obtain efficient (1)H spin locking and (1)H-(13)C decoupling at ultrafast MAS. As a result, large amounts of (1)H magnetization, from 35% to 42% of the initial polarization, are preserved after cross-polarization and decoupling. Restoring this magnetization to the longitudinal axis using a flip-back pulse leads to an enhancement of the sensitivity, an increase ranging from 14% to 21% in the maximal achievable sensitivity regime and from 24% to 50% in the fast pulsing regime, and to a shortening of the optimal recycling delay to 68% of its original duration. The analysis of the recovery and sensitivity curves reveals that the sensitivity gains do not rely on a selective transfer where few protons contribute but rather on careful conservation of bulk (1)H magnetization. This makes our method compatible with broadband experiments and uniformly labeled materials, in contrast to the enhancement schemes proposed for low MAS. We tested seven different cross-polarization schemes and determined that recovery of bulk (1)H magnetization is a general method for sensitivity enhancement. The physical insight gained about the behavior of proton magnetization sharing under spin lock will be helpful to break further sensitivity boundaries, when even higher external magnetic fields and faster spinning rates are employed.

Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.

Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively (13)C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy.

We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.

A straightforward method for stereospecific assignment of val and leu prochiral methyl groups by solid-state NMR: Scrambling in the [2-13C]Glucose labeling scheme.

The unambiguous stereospecific assignment of the prochiral methyl groups in Val and Leu plays an important role in the structural investigation of proteins by NMR. Here, we present a straightforward method for their stereospecific solid-state NMR assignment based on [2-(13)C]Glucose ([2-(13)C]Glc) as the sole carbon source during protein expression. The approach is fundamentally based on the stereo-selective biosynthetic pathway of Val and Leu, and the co-presence of [2-(13)C]pyruvate produced mainly by glycolysis and [3-(13)C]/[1,3-(13)C]pyruvate most probably formed through scrambling in the pentose phosphate pathway. As a consequence, the isotope spin pairs (13)Cß-(13)Cγ2 and (13)Cα-(13)Cγ1 in Val, and (13)Cγ-(13)Cδ2 and (13)Cß-(13)Cδ1 in Leu are obtained. The approach is successfully demonstrated with the stereospecific assignment of the methyl groups of Val and Leu of type 3 secretion system PrgI needles and microcrystalline ubiquitin.

Lewis base mediated dismutation of trichlorosilane.

An abnormal N-heterocyclic carbene (aNHC) has been used as a Lewis base to initiate dismutation of trichlorosilane. This report presents the reactivity differences of a normal N-heterocyclic carbene (NHC) versus aNHC with heavier group 14 elements. Three novel compounds (NHC)(2)·SiCl(2)H(2) (2), aNHC·SiCl(2)H(2) (3), and aNHC·GeCl(2) (4) have been synthesized and characterized by single crystal X-ray analysis, solid-state NMR and DFT calculations.

ß-Barrel mobility underlies closure of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is the major protein in the outer mitochondrial membrane, where it mediates transport of ATP and ADP. Changes in its permeability, induced by voltage or apoptosis-related proteins, have been implicated in apoptotic pathways. The three-dimensional structure of VDAC has recently been determined as a 19-stranded ß-barrel with an in-lying N-terminal helix. However, its gating mechanism is still unclear. Using solid-state NMR spectroscopy, molecular dynamics simulations, and electrophysiology, we show that deletion of the rigid N-terminal helix sharply increases overall motion in VDAC's ß-barrel, resulting in elliptic, semicollapsed barrel shapes. These states quantitatively reproduce conductance and selectivity of the closed VDAC conformation. Mutation of the N-terminal helix leads to a phenotype intermediate to the open and closed states. These data suggest that the N-terminal helix controls entry into elliptic ß-barrel states which underlie VDAC closure. Our results also indicate that ß-barrel channels are intrinsically flexible.

Monomeric Sn(II) and Ge(II) hydrides supported by a tridentate pincer-based ligand.

Herein we report the syntheses of terminal Sn(II) (3) and Ge(II) (4) hydrides from the corresponding chloride precursors [{2,6-iPr(2)C(6)H(3)NCMe}(2)C(6)H(3)MCl] (M = Sn (1), Ge (2)) using [K{B(sec-Bu)(3)}H] as a hydrogenating agent. Combination of steric shielding and intramolecular N → M interactions resulted in the protection of M(II)-H bonds.