Characterizing ATP processing by the AAA+ protein p97 at the atomic level.

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

Amyloid fibril structure from the vascular variant of systemic AA amyloidosis.

Systemic AA amyloidosis is a debilitating protein misfolding disease in humans and animals. In humans, it occurs in two variants that are called 'vascular' and 'glomerular', depending on the main amyloid deposition site in the kidneys. Using cryo electron microscopy, we here show the amyloid fibril structure underlying the vascular disease variant. Fibrils purified from the tissue of such patients are mainly left-hand twisted and contain two non-equal stacks of fibril proteins. They contrast in these properties to the fibrils from the glomerular disease variant which are right-hand twisted and consist of two structurally equal stacks of fibril proteins. Our data demonstrate that the different disease variants in systemic AA amyloidosis are associated with different fibril morphologies.

Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding.

Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.

Molecular mechanism of amyloidogenic mutations in hypervariable regions of antibody light chains.

Systemic light chain (AL) amyloidosis is a fatal protein misfolding disease in which excessive secretion, misfolding, and subsequent aggregation of free antibody light chains eventually lead to deposition of amyloid plaques in various organs. Patient-specific mutations in the antibody VL domain are closely linked to the disease, but the molecular mechanisms by which certain mutations induce misfolding and amyloid aggregation of antibody domains are still poorly understood. Here, we compare a patient VL domain with its nonamyloidogenic germline counterpart and show that, out of the five mutations present, two of them strongly destabilize the protein and induce amyloid fibril formation. Surprisingly, the decisive, disease-causing mutations are located in the highly variable complementarity determining regions (CDRs) but exhibit a strong impact on the dynamics of conserved core regions of the patient VL domain. This effect seems to be based on a deviation from the canonical CDR structures of CDR2 and CDR3 induced by the substitutions. The amyloid-driving mutations are not necessarily involved in propagating fibril formation by providing specific side chain interactions within the fibril structure. Rather, they destabilize the VL domain in a specific way, increasing the dynamics of framework regions, which can then change their conformation to form the fibril core. These findings reveal unexpected influences of CDR-framework interactions on antibody architecture, stability, and amyloid propensity.

The Binding Mode of the Sonic Hedgehog Inhibitor Robotnikinin, a Combined Docking and QM/MM MD Study.

Erroneous activation of the Hedgehog pathway has been linked to a great amount of cancerous diseases and therefore a large number of studies aiming at its inhibition have been carried out. One leverage point for novel therapeutic strategies targeting the proteins involved, is the prevention of complex formation between the extracellular signaling protein Sonic Hedgehog and the transmembrane protein Patched 1. In 2009 robotnikinin, a small molecule capable of binding to and inhibiting the activity of Sonic Hedgehog has been identified, however in the absence of X-ray structures of the Sonic Hedgehog-robotnikinin complex, the binding mode of this inhibitor remains unknown. In order to aid with the identification of novel Sonic Hedgehog inhibitors, the presented investigation elucidates the binding mode of robotnikinin by performing an extensive docking study, including subsequent molecular mechanical as well as quantum mechanical/molecular mechanical molecular dynamics simulations. The attained configurations enabled the identification of a number of key protein-ligand interactions, aiding complex formation and providing stabilizing contributions to the binding of the ligand. The predicted structure of the Sonic Hedgehog-robotnikinin complex is provided via a PDB file as Supplementary Material and can be used for further reference.