Two Bacterial Small Heat Shock Proteins, IbpA and IbpB, Form a Functional Heterodimer

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones which in stress conditions bind to unfolded protein substrates and prevent their irreversible aggregation. Substrates trapped in sHsps-containing aggregates are efficiently refolded into native structures by ATP-dependent Hsp70 and Hsp100 chaperones. Most γ-proteobacteria possess a single sHsp (IbpA), while in a subset of Enterobacterales, as a consequence of ibpA gene duplication event, a two-protein sHsp (IbpA and IbpB) system has evolved. IbpA and IbpB are functionally divergent. Purified IbpA, but not IbpB, stably interacts with aggregated substrates, yet both sHsps are required to be present at the substrate denaturation step for subsequent efficient Hsp70-Hsp100-dependent substrate refolding. IbpA and IbpB interact with each other, influence each other's expression levels and degradation rates. However, the crucial information on how these two sHsps interact and what is the basic building block required for proper sHsps functioning was missing. Here, based on NMR, mass spectrometry and crosslinking studies, we show that IbpA-IbpB heterodimer is a dominating functional unit of the two sHsp system in Enterobacterales. The principle of heterodimer formation is similar to one described for homodimers of single bacterial sHsps. β-hairpins formed by strands β5 and β7 of IbpA or IbpB crystallin domains associate with the other one's β-sandwich in the heterodimer structure. Relying on crosslinking and molecular dynamics studies, we also propose the orientation of two IbpA-IbpB heterodimers in a higher order tetrameric structure

Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates.

Small heat shock proteins (sHsps) are a conserved class of ATP-independent chaperones that bind to aggregation-prone polypeptides at stress conditions. sHsps encage these polypeptides in assemblies, shielding them from further aggregation. To facilitate their subsequent solubilization and refolding by Hsp70 (DnaK) and Hsp100 (ClpB) chaperones, first, sHsps need to dissociate from the assemblies. In most γ-proteobacteria, these functions are fulfilled by a single sHsp (IbpA), but in a subset of Enterobacterales, a two-protein sHsp (IbpA and IbpB) system has evolved. To gain insight into the emergence of complexity within this chaperone system, we reconstructed the phylogeny of γ-proteobacteria and their sHsps. We selected proteins representative of systems comprising either one or two sHsps and analysed their ability to form sHsps-substrate assemblies. All the tested IbpA proteins, but not IbpBs, stably interact with an aggregating substrate. Moreover, in Escherichia coli cells, ibpA but not ibpB suppress the growth defect associated with low DnaK level, which points to the major protective role of IbpA during the breakdown of protein quality control. We also examined how sHsps affect the association of Hsp70 with the assemblies at the initial phase of disaggregation and how they affect protein recovery after stress. Our results suggest that a single gene duplication event has given rise to the sHsp system consisting of a strong canonical binder, IbpA, and its non-canonical paralog IbpB that enhances sHsps dissociation from the assemblies. The cooperation between the sHsps reduces the demand for Hsp70 needed to outcompete them from the assemblies by promoting sHsps dissociation without compromising assembly formation at heat shock. This potentially increases the robustness and elasticity of sHsps protection against irreversible aggregation

Molecular Recognition in Complexes of TRF Proteins with Telomeric DNA

<div><p>Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD) to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions.</p></div

Direct and water-mediated hydrogen bonds between the TRF proteins and DNA bases.

<p>The pattern of direct and water-mediated hydrogen bonds between the TRF proteins and DNA bases. Only interactions between amino acid residues and nucleic bases are considered, as these base-specific contacts are potentially critical for sequence recognition. Filled circles at individual bases are scaled to reflect the probability that two residues are connected through a direct (orange) or water-mediated (cyan) hydrogen bond (for numeric probability values and hydrogen bond lifetimes, see Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089460#pone.0089460.s009" target="_blank">File S1</a> and Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089460#pone.0089460.s009" target="_blank">File S1</a>). All contacts that are made with probability 0.05 are included. Grey percentage bars show the probability for a given base to be involved, at any given time, in either direct or water-mediated hydrogen bonds with the protein.</p

Solvent accessible surface of TRFs as a function of their distance from the DNA axis.

<p>Hydrophobic (top), hydrophilic (middle) and overall (bottom) solvent accessible surface area of TRF1 and TRF2 as a function of the xy-distance between the protein and the DNA dodecamer.</p

Interaction energy profiles for individual protein residues.

<p>Interaction energy (computed as a sum of electrostatic and van der Waals contributions) between the charged amino acid residues and their surroundings (DNA and solvent combined) as a function of the distance between the protein and the DNA axis. The protein structure is subdivided into three separate regions: the N-terminal linker (top), the middle region (middle) and the C-terminal helix (bottom). For a full set of amino acid interaction energies, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089460#pone.0089460.s007" target="_blank">Fig. S7</a>.</p

Orientations of TRF1/TRF2 DBDs with respect to DNA as a function of xy-distance.

<p>Two-dimensional free energy surfaces for the xy-distance and the three rigid-body rotation angles defining the orientation of TRF1 and TRF2 DBD domains with respect to the DNA dodecamer (aligned with the z axis). To calculate the angles, we first obtained a rotation matrix which gives the best-fit of the instantaneous protein structure to the initial one in the tightly-bound complex with DNA. The Rotation matrix was then expressed as a product of three matrices representing rotations about the x, y and z axis and the rotation angles were derived from these matrices.</p

Protein entropy changes upon DNA binding.

<p>Conformational entropy per atom for individual protein residues in the tightly-bound complex with telomeric DNA (narrow bars) and in the isolated state (wide bars, colored according to amino acid type), estimated using the quasi-harmonic approximation. Only the regions interacting directly with DNA in the tightly-bound complex are presented: the N-terminal linker (purple) and the C-terminal helix (green).</p

Hydrogen bond profiles for individual TRF residues.

<p>The probability of hydrogen bond formation between individual protein residues and DNA as a function of the distance from the DNA axis. Only the residues for which the probability exceeds 0.2 are presented. The protein structure is subdivided into three separate regions: the N-terminal linker (top), the middle region (middle) and the C-terminal helix (bottom).</p

Detailed view of the interface between TRFs and DNA in the sequence-specific complex.

<p>Four amino acid residues important for recognition of telomeric dsDNA are shown explicitly. See legend to Fig. 1A for base color-coding.</p