The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery.

Investigating ligand-regulated allosteric coupling between protein domains is fundamental to understand cell-life regulation. The Hsp70 family of chaperones represents an example of proteins in which ATP binding and hydrolysis at the Nucleotide Binding Domain (NBD) modulate substrate recognition at the Substrate Binding Domain (SBD). Herein, a comparative analysis of an allosteric (Hsp70-DnaK) and a non-allosteric structural homolog (Hsp110-Sse1) of the Hsp70 family is carried out through molecular dynamics simulations, starting from different conformations and ligand-states. Analysis of ligand-dependent modulation of internal fluctuations and local deformation patterns highlights the structural and dynamical changes occurring at residue level upon ATP-ADP exchange, which are connected to the conformational transition between closed and open structures. By identifying the dynamically responsive protein regions and specific cross-domain hydrogen-bonding patterns that differentiate Hsp70 from Hsp110 as a function of the nucleotide, we propose a molecular mechanism for the allosteric signal propagation of the ATP-encoded conformational signal.

DnaK, the bacterial homolog of human Hsp70, plays an important role in pathogens survival under stress conditions, like antibiotic therapies. This chaperone sequesters protein aggregates accumulated in bacteria during antibiotic treatment reducing the effect of the cure. Although different classes of DnaK inhibitors have been already designed, they present low specificity. DnaK is highly conserved in prokaryotes (identity 50–70%), which encourages the development of a unique inhibitor for many different bacterial strains. We used the DnaK of Acinetobacter baumannii as representative for our analysis, since it is one of the most important opportunistic human pathogens, exhibits a significant drug resistance and it has the ability to survive in hospital environments. The E.coli DnaK was also included in the analysis as reference structure due to its wide diffusion. Unfortunately, bacterial DnaK and human Hsp70 have an elevated sequence similarity. Therefore, we performed a differential analysis of DnaK and Hsp70 residues to identify hot spots in bacterial proteins that are not present in the human homolog, with the aim of characterizing the key pharmacological features necessary to design selective inhibitors for DnaK. Different conformations of DnaK and Hsp70 bound to known inhibitor-peptides for DnaK, and ineffective for Hsp70, have been analysed by molecular dynamics simulations to identify residues displaying stable and selective interactions with these peptides. Results achieved in this work show that there are some residues that can be used to build selective inhibitors for DnaK, which should be ineffective for the human Hsp70.

Oxidative stress is a particularly severe threat to Antarctic marine polar organisms because they are exposed to high dissolved oxygen and to intense UV radiation. This paper reports the features of three superoxide dismutases from the Antarctic psychrophilic ciliate Euplotes focardii that faces two environmental challenges, oxidative stress and low temperature. Two out of these are Cu,Zn superoxide dismutases (named Ef-SOD1a and Ef-SOD1b) and one belongs to the Mn-containing group (Ef-SOD2). Ef-SOD1s and Ef-SOD2 differ in their evolutionary history, expression and overall structural features. Ef-SOD1 genes are expressed at different levels, with Ef-SOD1b mRNA 20-fold higher at the ciliate optimal temperature of growth (4 °C). All Ef-SOD enzymes are active at 4 °C, consistent with the definition of cold-adapted enzymes. At the same time, they display temperatures of melting in the range 50–70 °C and retain residual activity after incubation at 65–75 °C. Supported by data of molecular dynamics simulation, we conclude that the E. focardii SODs combine cold activity, local molecular flexibility and thermo tolerance.

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Cloud computing opens new perspectives for small-medium biotechnology laboratories that need to perform bioinformatics analysis in a flexible and effective way. This seems particularly true for hybrid clouds that couple the scalability offered by general-purpose public clouds with the greater control and ad hoc customizations supplied by the private ones. A hybrid cloud broker, acting as an intermediary between users and public providers, can support customers in the selection of the most suitable offers, optionally adding the provisioning of dedicated services with higher levels of quality. This paper analyses some economic and practical aspects of exploiting cloud computing in a real research scenario for the in silico drug discovery in terms of requirements, costs, and computational load based on the number of expected users. In particular, our work is aimed at supporting both the researchers and the cloud broker delivering an IaaS cloud infrastructure for biotechnology laboratories exposing different levels of nonfunctional requirements.

Hsp70 chaperones play important roles in cells including protein folding, trafficking, degradation and enabling survival under stress conditions. DnaK is an E. coli Hsp70 homolog comprising an ATPase domain and a substrate-binding domain. DnaK has two substrate-affinity states: ATP binding lowers the affinity of the substrate, whereas its hydrolysis leads to a higher affinity of substrate for binding. ATP-dependent communication between the two domains is essential for chaperone function and mediated via a conserved hydrophobic linker ( 384 GDVKDVLLL 392 ). Previous studies showed that when the linker interacts with the ATPase domain, which was studied by the construct containing the entire linker, DnaK(1-392), an enhanced ATPase rate is observed compared to the construct lacking the conserved 389 VLLL 392 linker region, DnaK(1-388). This observation suggests that structural rearrangements caused by linker docking adopt the ATPase domain in a closed conformation, leading to an enhanced, pH-dependent ATPase activity. Here, our aim is to delineate the residues that are responsible for the linker induced conformational rearrangements. In that line, using molecular dynamic simulations we identified a putative network of interactions through Arg71-Glu81-Asp85-Thr225-His226 at the lobe interface of the ATPase domain that might be critical in stabilization of the domain in the so called ''open'' and ''closed'' conformational equilibrium. We made point mutations for these sites on the two ATPase domain constructs, and studied the structural and functional effects of these residues on the ATPase domain as a function of pH using various biophysical and biochemical methods. Mutations' effects studied by equilibrium thermodynamic measurements showed variations for the constructs, but dramatic changes were observed in the dynamics of the constructs. Our results suggest the linker as a controller for ATPase domain dynamics changes partly through the identified network.

We identified two ice-binding protein (IBP) sequences, named EFsymbAFP and EFsymbIBP, from a putative bacterial symbiont of the Antarctic psychrophilic ciliate Euplotes focardii. EFsymbAFP is 57.43% identical to the antifreeze protein (AFP) from the Stigmatella aurantiaca strain DW4/3-1, which was isolated from the Victoria Valley lower glacier. EFsymbIBP is 53.38% identical to the IBP from the Flavobacteriaceae bacterium strain 3519-10, isolated from the glacial ice of Lake Vostok. EFsymbAFP and EFsymbIBP are 31.73% identical at the amino acid level and are organized in tandem on the bacterial chromosome. The relatively low sequence identity and the tandem organization, which appears unique to this symbiont, suggest an occurrence of horizontal gene transfer (HGT). Structurally, EFsymbAFP and EFsymbIBP are similar to the AFPs from the snow mould fungus Typhula ishikariensis and from the Arctic yeast Leucosporidium sp. AY30. A phylogenetic analysis showed that EFsymbAFP and EFsymbIBP cluster principally with the IBP sequences from other Antarctic bacteria, supporting the view that these sequences belong to an Antarctic symbiontic bacterium of E. focardii. These results confirm that IBPs have a complex evolutionary history, which includes HGT events, most probably due to the demands of the environment and the need for rapid adaptation.