Resolving the paradox for protein aggregation diseases: NMR structure and dynamics of the membrane-embedded P56S-MSP causing ALS imply a common mechanism for aggregation-prone proteins to attack membranes

Abstract

Paradoxically, aggregation of specific proteins is characteristic of many human diseases and aging, yet aggregates have increasingly been found to be unnecessary for initiating pathogenesis. Here we determined the NMR topology and dynamics of a helical mutant in a membrane environment transformed from the 125-residue cytosolic all-β MSP domain of vesicle-associated membrane protein-associated protein B (VAPB) by the ALS-causing P56S mutation. Despite its low hydrophobicity, the P56S major sperm protein (MSP) domain becomes largely embedded in the membrane environment with high backbone rigidity. Furthermore it is composed of five helices with amphiphilicity comparable to those of the partly-soluble membrane toxin mellitin and α-synuclein causing Parkinson's disease. Consequently, the mechanism underlying this chameleon transformation becomes clear: by disrupting the specific tertiary interaction network stabilizing the native all-β MSP fold to release previously-locked amphiphilic segments, the P56S mutation acts to convert the classic MSP fold into a membrane-active protein that is fundamentally indistinguishable from mellitin and α-synuclein which are disordered in aqueous solution but spontaneously partition into membrane interfaces driven by hydrogen-bond energetics gained from forming α-helix in the membrane environments. As segments with high amphiphilicity exist in all proteins, our study successfully resolves the paradox by deciphering that the proteins with a higher tendency to aggregate have a stronger potential to partition into membranes through the same mechanism as α-synuclein to initially attack membranes to trigger pathogenesis without needing aggregates. This might represent the common first step for various kinds of aggregated proteins to trigger familiar, sporadic and aging diseases. Therefore the homeostasis of aggregated proteins in vivo is the central factor responsible for a variety of human diseases including aging. The number and degree of the membrane attacks by aggregated proteins may act as an endogenous clock to count down the aging process. Consequently, a key approach to fight against them is to develop strategies and agents to maintain or even enhance the functions of the degradation machineries.

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

“…As the NS3pro contains no free Cys residue, three single-Cys mutants were prepared: Q27C, E86C, S158C by use of the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) as previously described [18]. The mutated plasmids were confirmed by DNA sequencing and their recombinant proteins were subsequently expressed and purified by the same procedures described above.…”

“…However, due to its "complete insolubility", it has been previously impossible to carry out any experimental studies on the isolated NS3pro domain. In 2005, we discovered that previously-thought "insoluble proteins" in fact could be solubilized in water with minimized salt ions [15][16][17][18], and therefore we have used this to study various previously-thought insoluble proteins including TDP-43 N-terminus [19]. Here with this approach, we have successfully characterized the solution conformations and dynamics of the isolated protease domain by CD, NMR and paramagnetic relaxation enhancement (PRE).…”

Environment-transformable sequence–structure relationship: a general mechanism for proteotoxicity

“…The formation of helix/loop results in the establishment of a large number of hydrogen bonds, thus leading to gain of hydrogen bond energetics (White and Wimley 1999;Ladokhin and White 1999), which acts to drive the partitioning from aqueous solutions into membrane environments. Therefore, the driving forces for a protein to aggregate and to partition into membrane environments appear to be at least partly overlapped, or even two sides of the same coin as previously proposed (Qin et al 2013b;Lim et al 2015;Lim and Song 2016;Song 2017).…”

“…Indeed, the disordered P56S MSP and its splicing variant did interact with the DPC micelle (Qin et al 2013a), as well as with the DMPC/ DHPC bicelle and DMPC vesicle mimicking the membrane environment (Qin et al 2013b). Subsequently, we determined the three-dimensional structure of P56S-MSP in the DPC micelle by a large set of NMR constraints, together with long-distance constraints derived from the paramagnetic relaxation enhancement.…”

“…Additionally, the eukaryotic, particularly human genomes appear to contain a large amount of IIPs, most, if not all, of which are also capable of interacting with membranes. Consequently, they might also act to initiate diseases and aging when their expression is increased, and/or degradation is reduced under some pathological, aging and/or environmental conditions (Nachreiner et al 2010;Qin et al 2013b).…”