Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

Detection and Alignment of 3D Domain Swapping Proteins Using Angle-Distance Image-Based Secondary Structural Matching Techniques

This work presents a novel detection method for three-dimensional domain swapping (DS), a mechanism for forming protein quaternary structures that can be visualized as if monomers had “opened” their “closed” structures and exchanged the opened portion to form intertwined oligomers. Since the first report of DS in the mid 1990s, an increasing number of identified cases has led to the postulation that DS might occur in a protein with an unconstrained terminus under appropriate conditions. DS may play important roles in the molecular evolution and functional regulation of proteins and the formation of depositions in Alzheimer's and prion diseases. Moreover, it is promising for designing auto-assembling biomaterials. Despite the increasing interest in DS, related bioinformatics methods are rarely available. Owing to a dramatic conformational difference between the monomeric/closed and oligomeric/open forms, conventional structural comparison methods are inadequate for detecting DS. Hence, there is also a lack of comprehensive datasets for studying DS. Based on angle-distance (A-D) image transformations of secondary structural elements (SSEs), specific patterns within A-D images can be recognized and classified for structural similarities. In this work, a matching algorithm to extract corresponding SSE pairs from A-D images and a novel DS score have been designed and demonstrated to be applicable to the detection of DS relationships. The Matthews correlation coefficient (MCC) and sensitivity of the proposed DS-detecting method were higher than 0.81 even when the sequence identities of the proteins examined were lower than 10%. On average, the alignment percentage and root-mean-square distance (RMSD) computed by the proposed method were 90% and 1.8Å for a set of 1,211 DS-related pairs of proteins. The performances of structural alignments remain high and stable for DS-related homologs with less than 10% sequence identities. In addition, the quality of its hinge loop determination is comparable to that of manual inspection. This method has been implemented as a web-based tool, which requires two protein structures as the input and then the type and/or existence of DS relationships between the input structures are determined according to the A-D image-based structural alignments and the DS score. The proposed method is expected to trigger large-scale studies of this interesting structural phenomenon and facilitate related applications

Folding mechanism of Pseudomonas aeruginosa cytochrome c551: role of electrostatic interactions on the hydrophobic collapse and transition state properties.

We report on the folding kinetics of the small 82 residue cytochrome c551from Pseudomonas aeruginosa. The presence of two Trp residues (Trp56 and Trp77) allows the monitoring of fluorescence quenching on refolding in two different regions of the protein. A single His residue (the iron-coordinating His16) permits the study of refolding in the absence of miscoordination events. After identification of the kinetic traps (Pro isomerization and aggregation of denatured protein), overall refolding kinetics is described by two processes: (i) a burstphase collapse (faster than milliseconds) which we show to be a global event leading to a state whose compactness depends on the overall net charge; at the isoeletric pH (4.7), it is maximally compact, while above and below it is more expanded; and (ii) an exponential phase (in the millisecond time range) leading to the native protein via a transition state(s) possibly involving the formation of a specific salt bridge between Lys10 and Glu70, at the contact between the N and C-terminal helices. Comparison with the widely studied horse cytochrome c allows the discussion of similarities and differences in the folding of two proteins which have the same "fold" despite a very low degree of sequence homology (<30 %)

Equilibrium unfolding of a small bacterial cytochrome, cytochrome c551 from Pseudomonas aeruginosa.

The unfolding of the small cytochrome c551 from the bacterium Pseudomonas aeruginosa has been characterized at equilibrium by circular dichroism (CD) and fluorescence spectroscopy. The process can be described by a two state mechanism and the thermodynamic stability of cytochrome c551 is found to be smaller than that of the larger horse cytochrome c (deltaGw = -8.2 vs. -9.7 kcal/mol); we propose that this finding is related to the absence of an 'omega' loop in the bacterial cytochrome. Cytochrome c551 loses most of its secondary structure at pH 1.5. The acid transition (pKA approximately 2) is highly cooperative (n > or =2); analysis of optical titrations and contact map suggests that (at least) His-16 (proximal Fe3+ ligand) and Glu-70 are both involved in the acid transition. The role of selected hydrophobic, electrostatic and conformational contributions to the overall stability has been investigated by protein engineering. The equilibrium characterization of wild-type and mutant cytochrome c551 supports the view that this small cytochrome is an interesting protein to analyze the thermodynamics and the kinetics of folding in comparison with the widely studied horse cytochrome c