This paper aims to understand the binding strategies of a nanobody-protein pair by studying known complexes. Rigid body protein-ligand docking programs produce several complexes, called decoys, which are good candidates with high scores of shape complementarity, electrostatic interactions, desolvation, buried surface area, and Lennard-Jones potentials. However, the decoy that corresponds to the native structure is not known. We studied 36 nanobody-protein complexes from the single domain antibody database, sd-Ab DB, http://www.sdab-db.ca/. For each structure, a large number of decoys are generated using the Fast Fourier Transform algorithm of the software ZDOCK. The decoys were ranked according to their target protein-nanobody interaction energies, calculated by using the Dreiding Force Field, with rank 1 having the lowest interaction energy. Out of 36 protein data bank (PDB) structures, 25 true structures were predicted as rank 1. Eleven of the remaining structures required Ångstrom size rigid body translations of the nanobody relative to the protein to match the given PDB structure. After the translation, the Dreiding interaction (DI) energies of all complexes decreased and became rank 1. In one case, rigid body rotations as well as translations of the nanobody were required for matching the crystal structure. We used a Monte Carlo algorithm that randomly translates and rotates the nanobody of a decoy and calculates the DI energy. Results show that rigid body translations and the DI energy are sufficient for determining the correct binding location and pose of ZDOCK created decoys. A survey of the sd-Ab DB showed that each nanobody makes at least one salt bridge with its partner protein, indicating that salt bridge formation is an essential strategy in nanobody-protein recognition. Based on the analysis of the 36 crystal structures and evidence from existing literature, we propose a set of principles that could be used in the design of nanobodies.

In enteropathogen, Yersinia enterocolitica, the genes encoding phage shock proteins are organized in an operon (pspA-E), which is activated at the various types of cellular stress (i.e., extracytoplasmic or envelop stress) whereas, PspA negatively regulates PspF, a transcriptional activator of pspA-E and pspG, and is also involved in other cellular machinery maintenance processes. The exact mechanism of association and dissociation of PspA and PspF during the stress response is not entirely clear. In this concern, we address conformational change of PspA in different pH conditions using various in-silico and biophysical methods. At the near-neutral pH, CD and FTIR measurements reveal a ß-like conformational change of PspA; however, AFM measurement indicates the lower oligomeric form at the above-mentioned pH. Additionally, the results of the MD simulation also support the conformational changes which indicate salt-bridge strength takes an intermediate position compared to other pHs. Furthermore, the bio-layer interferometry study confirms the stable complex formation that takes place between PspA and PspF at the near-neutral pH. It, thus, appears that PspA conformational change in adverse pH conditions abandons PspF from having a stable complex with it, and thus, the latter can act as a trans-activator. Taken together, it seems that PspA alone can transduce adverse signals by changing its conformation.

Salt-bridges (sb) play an important role in the folding and stability of proteins. This is deduced from the evaluation of net energy in the microenvironments (ME, residues that are 4 Å away from positive and negative partners of salt-bridge and interact with them). MEs act as a determinant of net-energy due to the intrinsic features in the sequence. The stability of extremophilic proteins is due to the presence of favorable residues at the ME without any unfavorable residues. We studied a dataset of four structures from the protein data bank (PDB) and a homology model (1HM5) to gain insights on this issue. Data shows that the presence of isolated charges and polar residues in the core of extremophilic proteins helps in the formation of stable salt-bridges with reduced desolvation. Thus, site-specific mutations with favorable residues at the ME will help to develop thermo stable proteins with strong salt bridges.

[This corrects the article DOI: 10.3389/fmolb.2020.00051.].

Limit dextrinase (LD) is the only endogenous starch-debranching enzyme in barley (Hordeum vulgare, Hv), which is the key factor affecting the production of a high degree of fermentation. Free LD will lose its activity in the mashing process at high temperature in beer production. However, there remains a lack of understanding on the factor affecting the themostability of HvLD at the atomic level. In this work, the molecular dynamics simulations were carried out for HvLD to explore the key factors affecting the thermal stability of LD. The higher value of root mean square deviation (RMSD), radius of gyration (R g), and surface accessibility (SASA) suggests the instability of HvLD at high temperatures. Intra-protein hydrogen bonds and hydrogen bonds between protein and water decrease at high temperature. Long-lived hydrogen bonds, salt bridges, and hydrophobic contacts are lost at high temperature. The salt bridge interaction analysis suggests that these salt bridges are important for the thermostability of HvLD, including E568-R875, D317-R378, D803-R884, D457-R214, D468-R395, D456-R452, D399-R471, and D541-R542. Root mean square fluctuation (RMSF) analysis identified the thermal-sensitive regions of HvLD, which will facilitate enzyme engineering of HvLD for enhanced themostability.

Salt-bridges play a unique role in the structural and functional stability of proteins, especially under harsh environments. How these salt-bridges contribute to the overall thermodynamic stability of protein structure and function across different domains of life is elusive still date. To address the issue, statistical analyses on the energies of salt-bridges, involved in proteins\' structure and function, are performed across three domains of life, that is, archaea, eubacteria, and eukarya. Results show that although the majority of salt-bridges are stable and conserved, yet the stability of archaeal proteins (∆∆Gnet = -5.06 ± 3.8) is much more than that of eubacteria (∆∆Gnet = -3.7 ± 2.9) and eukarya (∆∆Gnet = -3.54 ± 3.1). Unlike earlier study with archaea, in eukarya and eubacteria, not all buried salt-bridge in our dataset are stable. Buried salt-bridges play surprising role in protein stability, whose variations are clearly observed among these domains. Greater desolvation penalty of buried salt-bridges is compensated by stable network of salt-bridges apart from equal contribution of bridge and background energy terms. On the basis proteins\' secondary structure, topology, and evolution, our observation shows that salt-bridges when present closer to each other in sequence tend to form a greater number. Overall, our comparative study provides insight into the role of specific electrostatic interactions in proteins from different domains of life, which we hope, would be useful for protein engineering and bioinformatics study.

Extracellular plaques, the hallmark of Alzheimer\'s disease brains, consist of insoluble amyloid fibrils that result from the aggregation of amyloid beta peptides. None of the few therapeutic options currently adopted, address the cause of the disease. Instead, they reduce symptom of the disease. Inhibition of aggregation or destabilization of aggregates therefore, emerges as a preferable therapeutic approach. Designing inhibitors or destabilizers demands comprehensive knowledge of the residues of amyloid beta responsible for the phenomenal structural stability of the aggregate. For the purpose, we have compared the effect on structural destabilization of 13 in silico mutations (single and double) with the wild type counterpart of beta-strand-turn-beta-strand motif of the amyloid beta protofibrils by molecular dynamics simulation. Besides the already known salt bridge interaction between K28 and D23, our analyses expose more significant role of K28 as the only positive charge present in the vicinity. Amongst the two consecutive aromatic residues, F19 is involved in stacking interaction; although effect of F20 mutation is more pronounced. Face to face arrangement of A21 and V36 acts as a pillar maintaining the necessary optimum distance between consecutive chains to promote stabilizing interactions. In addition to providing stability to the first beta-strand, large sized negatively charged E22 facilitates salt bridge formation by ensuring fixed relative position of D23 and in turn K28. Likewise, the hydrophobic residues I32 and L34 pack the protofibril core, once again fostering salt bridge interaction. Prospectively, these findings may be compiled for efficient identification or design of scaffolds accountable for protofibril destabilization.Communicated by Ramaswamy H. Sarma.

Global minimal structure of protein/enzyme is energetically compromised that maintains an intricate balance between the rigidity and the flexibility. Such a state makes it interactive to its ligand molecules. Although protein data bank files (PDB) may have achieved the state, in many situations minimization has been crucial to overcome unwanted steric clashes, and other conformational strains. It is more so, when orthologous PDB structures that are intended in a given study, show variations in resolution, R-factor, shell-water contents, loop characteristics etc. Here, a fully automated procedure of minimization would be highly useful. AUTOMINv1.0 is such an automation of minimization that runs on any number of structure files with any number of chains in them along with the inclusion of selective/non-selective shell-waters interacting with polar and or non-polar atom-types of protein. Comparison of the mean binaryitems of salt-bridges of minimized and un-minimized structures (chains > 100) of nucleoside diphosphate kinase from mimi virus shows dramatic improvements in the earlier. Again, the mean steric clashes of 2AZ3.pdb are reduced upon minimization. Remarkably, the observed steric clashes between shell-waters and atom-types of protein are seen to be removed upon minimization. Taken together, AUTOMINv1.0 is an automation of minimization that finds applications in structural bioinformatics.

Zinc fingers are proteins that are characterized by the coordination of zinc ions by an amino acid sequence that commonly contains two histidines and two cysteines (2His-2Cys motif). Investigations of oligopeptides that contain the 2His-2Cys motif, e.g., acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, have discovered they exhibit pH-dependent Zn(II) chelation and have redox activities with Cu(I/II), forming a variety of metal complexes. To further understand how these 2His-2Cys oligopeptides bind these metal ions, we have undertaken a series of ion mobility-mass spectrometry and B3LYP/LanL2DZ computational studies of structurally related heptapeptides. Starting with the sequence above, we have modified the potential His, Cys, or C-terminus binding sites and report how these changes in primary structure affect the oligopeptides positive and negative charge states, conformational structure, collision-induced breakdown energies, and how effectively Zn(II) binds to these sequences. The results show evidence that the weak acid-base properties of Cys-His are intrinsically linked and can result in an intramolecular salt-bridged network that affects the oligopeptide properties.

A characteristic feature of higher-order structures of amyloid β peptide (Aβ) aggregates observed in Alzheimer disease is the salt-bridge between the side-chains of Asp23 (carboxylate) and Lys28 (ammonium). We synthesized an [Met35(O)]Aβ42 possessing a covalently bound lactam tether as an Asp23/Lys28 salt-bridge surrogate (compound 3). The lactam tether of 3 markedly promoted the formation of stable protofibril-like species that exhibited amyloidogenic properties such as a cross-β-sheet structure and cytotoxicity. This finding is consistent with reports that the Asp23/Lys28 salt-bridge of Aβ42 is transiently formed in aggregation intermediates.