Intrinsic Order and Disorder in the Bcl-2 Member Harakiri: Insights into Its Proapoptotic Activity

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-xL and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-xL and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works

Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study

Correction: Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study. PLoS ONE 9(1): 10.1371/annotation/dbb84118-9ada-43e4-8734-8f8322be1a59. doi: 10.1371/annotation/dbb84118-9ada-43e4-8734-8f8322be1a59Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.This work was financed by the Andalucía Government (grant FQM-02838), the Spanish Ministry of Science and Innovation (grant BIO2009-07317), and the European Regional Development Fund of the European Union. D. Ruzafa is recipient of a research fellowship from the F.P.U. program of the Spanish Ministry of Education. L. Varela is financed by the G.R.E.I.B. program of the University of Granada

Intrinsic Order and Disorder in the Bcl-2 Member Harakiri: Insights into Its Proapoptotic Activity

Harakiri is a BH3-only member of the Bcl-2 family that localizes in membranes and induces cell death by binding to prosurvival Bcl-xL and Bcl-2. The cytosolic domain of Harakiri is largely disorder with residual α-helical conformation according to previous structural studies. As these helical structures could play an important role in Harakiri's function, we have used NMR and circular dichroism to fully characterize them at the residue-atomic level. In addition, we report structural studies on a peptide fragment spanning Harakiri's C-terminal hydrophobic sequence, which potentially operates as a transmembrane domain. We initially checked by enzyme immunoassays and NMR that peptides encompassing different lengths of the cytosolic domain are functional as they bind Bcl-xL and Bcl-2. The structural data in water indicate that the α-helical conformation is restricted to a 25-residue segment comprising the BH3 domain. However, structure calculation was precluded because of insufficient NMR restraints. To bypass this problem we used alcohol-water mixture to increase structure population and confirmed by NMR that the conformation in both milieus is equivalent. The resulting three-dimensional structure closely resembles that of peptides encompassing the BH3 domain of BH3-only members in complex with their prosurvival partners, suggesting that preformed structural elements in the disordered protein are central to binding. In contrast, the transmembrane domain forms in micelles a monomeric α-helix with a population close to 100%. Its three-dimensional structure here reported reveals features that explain its function as membrane anchor. Altogether these results are used to propose a tentative structural model of how Harakiri works

Highly Anomalous Energetics of Protein Cold Denaturation Linked to Folding-Unfolding Kinetics

Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a “mirror image” of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions

Hydrogen-Bond Driven Loop-Closure Kinetics in Unfolded Polypeptide Chains

Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20–100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient β-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events