Recognizing centromere by kinetochore: The kinetochore proteins CENP-N and CENP-C recognize the histone H3 variant CENP-A in the centromeric nucleosome. This ensures proper kinetochore assembly and accurate segregation of chromosomes. Chittori et al. describe the cryo-electron microscopy structure of the human CENP-A nucleosome-CENP-N complex. The interaction of CENP-N with CENP-A and the nucleosomal DNA together ensure specific and stable centromeric nucleosome recognition. Mutational analyses using both human and Xenopus CENP-A and CENP-N proteins suggest that the proteins have coevolved to preserve the interacting surfaces. Science , this issue p. 339

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MPro^WT) was assessed using a predominantly monomeric mutant (MPro^M). Rates of MPro^WT and MPro^M catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MPro^WT, to MPro^M leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.

Significance: The root cause of pathology in sickle cell disease is the polymerization of the mutant hemoglobin S upon deoxygenation in the tissues to form fibers. Both the amount of fiber at equilibrium and the kinetics of fiber formation depend on the partial pressure of oxygen. We show that control of polymerization by oxygen at equilibrium can be better explained by a recent extension of the famous two-state allosteric model of Monod, Wyman, and Changeux. Because of the unusual kinetics, polymerization is far out of equilibrium, which explains why patients with the disease manage to survive, while those with high levels of fetal hemoglobin and those with sickle trait (the heterozygous condition) have relatively benign conditions.

Heptad repeat regions of SARS-CoV-2 spike protein transiently disrupt membrane integrity while driving cellular entry.

The monomeric catalytic domain (residues 1–199) of SARS-CoV-2 main protease (MPro^1-199) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro^1-199 junction. We report the catalytic activity and the dissociation constants of MPro^1-199 and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MPro^WT and MPro^1-199 and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.

Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.