Characterization of ßB2-crystallin tryptophan mutants reveals two different folding states in solution.

Conserved tryptophan residues are critical for the structure and the stability of ß/γ-crystallin in the lenses of vertebrates. During aging, in which the lenses are continuously exposed to ultraviolet irradiation and other environmental stresses, oxidation of tryptophan residues in ß/γ-crystallin is triggered and impacts the lens proteins to varying degrees. Kynurenine derivatives, formed by oxidation of tryptophan, accumulate, resulting in destabilization and insolubilization of ß/γ-crystallin, which correlates with age-related cataract formation. To understand the contribution of tryptophan modification on the structure and stability of human ßB2-crystallin, five tryptophan residues were mutated to phenylalanine considering its similarity in structure and hydrophilicity to kynurenine. Among all mutants, W59F and W151F altered the stability and homo-oligomerization of ßB2-crystallin-W59F promoted tetramerization whereas W151F blocked oligomerization. Most W59F dimers transformed into tetramer in a month, and the separated dimer and tetramer of W59F demonstrated different structures and hydrophobicity, implying that the biochemical properties of ßB2-crystallin vary over time. By using SAXS, we found that the dimer of ßB2-crystallin in solution resembled the lattice ßB1-crystallin dimer (face-en-face), whereas the tetramer of ßB2-crystallin in solution resembled its lattice tetramer (domain-swapped). Our results suggest that homo-oligomerization of ßB2-crystallin includes potential inter-subunit reactions, such as dissociation, unfolding, and re-formation of the dimers into a tetramer in solution. The W>F mutants are useful in studying different folding states of ßB2-crystallin in lens.

Structural basis for the allosteric behaviour and substrate specificity of Lactococcus lactis Prolidase.

Prolidase (EC 3.4.13.9) is an enzyme that specifically hydrolyzes Xaa-Pro dipeptides into free amino acids. We previously studied kinetic behaviours and solved the crystal structure of wild-type (WT) Lactococcus lactis prolidase (Llprol), showing that this homodimeric enzyme has unique characteristics: allosteric behaviour and substrate inhibition. In this study, we focused on solving the crystal structures of three Llprol mutants (D36S, H38S, and R293S) which behave differently in v-S plots. The D36S and R293S Llprol mutants do not show allosteric behaviour, and the Llprol mutant H38S has allosteric behaviour comparable to the WT enzyme (Hill constant 1.52 and 1.58, respectively). The crystal structures of Llprol variants suggest that the active site of Llprol formed with amino acid residues from both monomers, i.e., located in an interfacial area of dimer. The comparison between the structure models of Llprol indicated that the two monomers in the dimers of Llprol variants have different relative positions among Llprol variants. They showed different interatomic distances between the amino acid residues bridging the two monomers and varied sizes of the solvent-accessible interface areas in each Llprol variant. These observations indicated that Llprol could adapt to different conformational states with distinctive substrate affinities. It is strongly speculated that the domain movements required for productive substrate binding are restrained in allosteric Llprol (WT and H38S). At low substrate concentrations, only one out of the two active sites at the dimer interface could accept substrate; as a result, the asymmetrical activated dimer leads to allosteric behaviour.

Defective ryanodine receptor N-terminus inter-subunit interaction is a common mechanism in neuromuscular and cardiac disorders.

The ryanodine receptor (RyR) is a homotetrameric channel mediating sarcoplasmic reticulum Ca2+ release required for skeletal and cardiac muscle contraction. Mutations in RyR1 and RyR2 lead to life-threatening malignant hyperthermia episodes and ventricular tachycardia, respectively. In this brief report, we use chemical cross-linking to demonstrate that pathogenic RyR1 R163C and RyR2 R169Q mutations reduce N-terminus domain (NTD) tetramerization. Introduction of positively-charged residues (Q168R, M399R) in the NTD-NTD inter-subunit interface normalizes RyR2-R169Q NTD tetramerization. These results indicate that perturbation of NTD-NTD inter-subunit interactions is an underlying molecular mechanism in both RyR1 and RyR2 pathophysiology. Importantly, our data provide proof of concept that stabilization of this critical RyR1/2 structure-function parameter offers clear therapeutic potential.

Origin of Increased Solvent Accessibility of Peptide Bonds in Mutual Synergetic Folding Proteins.

Mutual Synergetic Folding (MSF) proteins belong to a recently discovered class of proteins. These proteins are disordered in their monomeric but ordered in their oligomeric forms. Their amino acid composition is more similar to globular proteins than to disordered ones. Our preceding work shed light on important structural aspects of the structural organization of these proteins, but the background of this behavior is still unknown. We suggest that solvent accessibility is an important factor, especially solvent accessibility of the peptide bonds can be accounted for this phenomenon. The side chains of the amino acids which form a peptide bond have a high local contribution to the shielding of the peptide bond from the solvent. During the oligomerization step, other non-local residues contribute to the shielding. We investigated these local and non-local effects of shielding based on Shannon information entropy calculations. We found that MSF and globular homodimeric proteins have different local contributions resulting from different amino acid pair frequencies. Their non-local distribution is also different because of distinctive inter-subunit contacts.

Physical Background of the Disordered Nature of "Mutual Synergetic Folding" Proteins.

Intrinsically disordered proteins (IDPs) lack a well-defined 3D structure. Their disordered nature enables them to interact with several other proteins and to fulfil their vital biological roles, in most cases after coupled folding and binding. In this paper, we analyze IDPs involved in a new mechanism, mutual synergistic folding (MSF). These proteins define a new subset of IDPs. Recently we collected information on these complexes and created the Mutual Folding Induced by Binding (MFIB) database. These protein complexes exhibit considerable structural variation, and almost half of them are homodimers, but there is a significant amount of heterodimers and various kinds of oligomers. In order to understand the basic background of the disordered character of the monomers found in MSF complexes, the simplest part of the MFIB database, the homodimers are analyzed here. We conclude that MFIB homodimeric proteins have a larger solvent-accessible main-chain surface area on the contact surface of the subunits, when compared to globular homodimeric proteins. The main driving force of the dimerization is the mutual shielding of the water-accessible backbones and the formation of extra intermolecular interactions.