No abstract

The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

HgammaC-Crys formed amyloid fibrils on incubation at low pH via a partially unfolded intermediate. This process could contribute to the early stages of the formation of light-scattering species in the eye lens.

The A-to-B form transition has been examined in three DNA duplexes, d(CGCGAATTCGCG)(2), d(CGCGAATTGCGC), and d(CGCAAATTTCGC), using circular dichroism spectroscopy, ultraviolet resonance Raman (UVRR) spectroscopy, and molecular dynamics (MD) simulation. Circular dichroism spectra confirm that these molecules adopt the A form under conditions of reduced water activity. UVRR results, obtained under similar conditions, suggest that the transition involves a series of intermediate forms between A and B. Cooperative and distinct transitions were observed for the bases and the sugars. Independent MD simulations on d(CGCGAATTCGCG)(2) show a spontaneous change from the A to B form in aqueous solution and describe a kinetic model that agrees well with UVRR results. Based on these observations, we predict that the mechanism of the transition involves a series of A/B hybrid forms and is sequential in nature, similar to previous crystallographic studies of derivatized duplexes. A simulation in which waters were restrained in the major groove of B DNA shows a rapid, spontaneous change from B to A at reduced water activity. These results indicate that a quasiergodic sampling of the solvent distribution may be a problem in going from B to A at reduced water activity in the course of an MD simulation.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (β6 Glu → Val) on the β-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.