Dynamic lipid interactions in the plasma membrane Na+,K+-ATPase.

The function of ion-transporting Na+,K+-ATPases depends on the surrounding lipid environment in biological membranes. Two established lipid-interaction sites A and B within the transmembrane domain have been observed to induce protein activation and stabilization, respectively. In addition, lipid-mediated inhibition has been assigned to a site C, but with the exact location not experimentally confirmed. Also, possible effects on lipid interactions by disease mutants dwelling in the membrane-protein interface remain relatively uncharacterized. We simulated human Na+,K+-ATPase α1ß1FXYD homology models in E1 and E2 states in an asymmetric, multicomponent plasma membrane to determine both wild-type and disease mutant lipid-protein interactions. The simulated wild-type lipid interactions at the established sites A and B were in agreement with experimental results thereby confirming the membrane-protein model system. The less well-characterized, proposed inhibitory site C was dominated by lipids lacking inhibitory properties. Instead, two sites hosting inhibitory lipids were identified at the extracellular side and also a cytoplasmic CHL-binding site that provide putative alternative locations of Na+,K+-ATPase inhibition. Three disease mutations, Leu302Arg, Glu840Arg and Met859Arg resided in the lipid-protein interface and caused drastic changes in the lipid interactions. The simulation results show that lipid interactions to the human Na+,K+-ATPase α1ß1FXYD protein in the plasma membrane are highly state-dependent and can be disturbed by disease mutations located in the lipid interface, which can open up for new venues to understand genetic disorders.

Natural furin inhibitor(s) as potent therapeutic molecule to mitigate SARS-CoV-2 infection.

The COVID 2019 has had been going pandemic as per WHO situation reports. The major differentiating point in this virus is the presence of a unique furin cleavage site. Our insilico study points out to the effectiveness of a potent plant origin furin inhibitor. We exploited the aspect of the cleavage machinery of furin subunits which is critical and indispensible to the entry of SARS-CoV-2 in human cells and subsequent massive contagion. In-silico analysis was done to observe the interactions of proposed analogs of protease inhibitor of plant origin against furin protein as well as the furin spike glycoprotein (SGP) binding machinery. This was done by docking protocols using Hex 6.0 software followed by molecular Dynamic simulation (MDS) analysis in 100 ns scale using Amber94. Further, the images were analysed with PyMol software. The analogs I, II and III included in our study showed strong interactivity against furin individually, as well as the furin-Spike Glycoprotein 1 binding machinery. The findings were confirmed using molecular dynamic simulation analysis which indicated good structural stability and ability to neutralise furin and furin-spike glycoprotein 1. Analog II was found to be the best interactive molecule against furin, showing the least deviation (1.484 ± 0.0064). Also, it was the most effective against furin + Spike glycoprotein I machinery [1.575 ± 0.01]. We report the first of its kind of natural furin inhibitor(s) which would disrupt the furin machinery of SARS-CoV-2 and help in controlling the COVID-19 contagion.Communicated by Ramaswamy H. Sarma.

Retracing the Rapid Evolution of an Herbicide-Degrading Enzyme by Protein Engineering.

The mechanisms underlying the rapid evolution of novel enzymatic activities from promiscuous side activities are poorly understood. Recently emerged enzymes catalyzing the catabolic degradation of xenobiotic substances that have been spread out into the environment during the last decades provide an exquisite opportunity to study these mechanisms. A prominent example is the herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine), which is degraded through a number of enzymatic reactions constituting the Atz pathway. Here, we analyzed the evolution of the hydroxyatrazine ethylaminohydrolase AtzB, a Zn(II)-dependent metalloenzyme that adopts the amidohydrolase fold and catalyzes the second step of the Atz pathway. We searched for promiscuous side activities of AtzB, which might point to the identity of its progenitor. These investigations revealed that AtzB has low promiscuous guanine deaminase activity. Furthermore, we found that the two closest AtzB homologues, which have not been functionally annotated up to now, are guanine deaminases with modest promiscuous hydroxyatrazine hydrolase activity. Based on sequence comparisons with the closest AtzB homologues, the guanine deaminase activity of AtzB could be increased by three orders of magnitude through the introduction of only four active site mutations. Interestingly, introducing the inverse four mutations into the AtzB homologues significantly enhanced their hydroxyatrazine hydrolase activity, and in one case is even equivalent to that of wild-type AtzB. Molecular dynamics simulations elucidated the structural and molecular basis for the mutation-induced activity changes. The example of AtzB highlights how novel enzymes with high catalytic proficiency can evolve from low promiscuous side activities by only few mutational events within a short period of time.

The two-domain elevator-type mechanism of zinc-transporting ZIP proteins.

Zinc is essential for all organisms and yet detrimental at elevated levels. Hence, homeostasis of this metal is tightly regulated. The Zrt/Irt-like proteins (ZIPs) represent the only zinc importers in metazoans. Mutations in human ZIPs cause serious disorders, but the mechanism by which ZIPs transfer zinc remains elusive. Hitherto, structural information is only available for a model member, BbZIP, and as a single, ion-bound conformation, precluding mechanistic insights. Here, we elucidate an inward-open metal-free BbZIP structure, differing substantially in the relative positions of the two separate domains of ZIPs. With accompanying coevolutional analyses, mutagenesis, and uptake assays, the data point to an elevator-type transport mechanism, likely shared within the ZIP family, unifying earlier functional data. Moreover, the structure reveals a previously unknown ninth transmembrane segment that is important for activity in vivo. Our findings outline the mechanistic principles governing ZIP-protein transport and enhance the molecular understanding of ZIP-related disorders.

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper.

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical ß-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

Structure and ion-release mechanism of PIB-4-type ATPases.

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal PIB-4-ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

Heavy metals such as zinc and cobalt are toxic at high levels, yet most organisms need tiny amounts for their cells to work properly. As a result, proteins studded through the cell membrane act as gatekeepers to finetune import and export. These proteins are central to health and disease; their defect can lead to fatal illnesses in humans, and they also help bacteria infect other organisms. Despite their importance, little is known about some of these metal-export proteins. This is particularly the case for P_IB-4-ATPases, a subclass found in plants and bacteria and which includes, for example, a metal transporter required for bacteria to cause tuberculosis. Intricate knowledge of the three-dimensional structure of these proteins would help to understand how they select metals, shuttle the compounds in and out of cells, and are controlled by other cellular processes. To reveal this three-dimensional organisation, Grønberg et al. used X-ray diffraction, where high-energy radiation is passed through crystals of protein to reveal the positions of atoms. They focused on a type of PIB-4-ATPases found in bacteria as an example. The work showed that the protein does not contain the metal-binding regions seen in other classes of metal exporters; however, it sports unique features that are crucial for metal transport such as an adapted pathway for the transport of zinc and cobalt across the membrane. In addition, Grønberg et al. tested thousands of compounds to see if they could block the activity of the protein, identifying two that could kill bacteria. This better understanding of how PIB-4-ATPases work could help to engineer plants capable of removing heavy metals from contaminated soils, as well as uncover new compounds to be used as antibiotics.

Bioengineering of non-pathogenic Escherichia coli to enrich for accumulation of environmental copper.

Heavy metal sequestration from industrial wastes and agricultural soils is a long-standing challenge. This is more critical for copper since copper pollution is hazardous both for the environment and for human health. In this study, we applied an integrated approach of Darwin's theory of natural selection with bacterial genetic engineering to generate a biological system with an application for the accumulation of Cu2+ ions. A library of recombinant non-pathogenic Escherichia coli strains was engineered to express seven potential Cu2+ binding peptides encoded by a 'synthetic degenerate' DNA motif and fused to Maltose Binding Protein (MBP). Most of these peptide-MBP chimeras conferred tolerance to high concentrations of copper sulphate, and in certain cases in the order of 160-fold higher than the recognised EC50 toxic levels of copper in soils. UV-Vis spectroscopic analysis indicated a molar ratio of peptide-copper complexes, while a combination of bioinformatics-based structure modelling, Cu2+ ion docking, and MD simulations of peptide-MBP chimeras corroborated the extent of Cu2+ binding among the peptides. Further, in silico analysis predicted the peptides possessed binding affinity toward a broad range of divalent metal ions. Thus, we report on an efficient, cost-effective, and environment-friendly prototype biological system that is potentially capable of copper bioaccumulation, and which could easily be adapted for the removal of other hazardous heavy metals or the bio-mining of rare metals.

Microscopic and spectroscopic characterization of rice and corn starch.

Starch granules from rice and corn were isolated, and their molecular mechanism on interaction with α-amylase was characterized through biochemical test, microscopic imaging, and spectroscopic measurements. The micro-scale structure of starch granules were observed under an optical microscope and their average size was in the range 1-100 µm. The surface topological structures of starch with micro-holes due to the effect of α- amylase were also visualized under scanning electron microscope. The crystallinity was confirmed by X-ray diffraction patterns as well as second-harmonic generation microscopy. The change in chemical bonds before and after hydrolysis of the starch granules by α- amylase was determined by Fourier transform infrared spectroscopy. Combination of microscopy and spectroscopy techniques relates structural and chemical features that explain starch enzymatic hydrolysis which will provide a valid basis for future studies in food science and insights into the energy transformation dynamics.

Specification of binding modes between a transmembrane peptide mimic of ATP6V0C and polytopic E5 of human papillomavirus-16.

Interaction of E5 of papillomavirus-16 based on its three transmembrane domains (TMDs) with a peptide mimicking the fourth TMD (TMD-A) of the 16 kDa c subunit of the human vacuolar H+-ATPase, ATP6V0C, and one of its mutant is investigated. Docking reveals binding of the peptide between the second and third TMD of E5. A series of hydrophobic residues are responsible for the contact. Estimated weak binding energies based on potential of mean force calculations reveal marginal differences of the estimated binding energies between wild type (WT) and mutant peptide. Also differences in estimated binding energies of dimers of the individual TMDs of E5 with the WT peptide are marginal. Correlation of rotational data derived from coarse-grained molecular dynamics simulations of the peptides and the protein as well as from the principal component analysis reveal that the binding of TMD-A with TMD3 is enthalpy driven and binding with TMD2 is guided by entropic conditions.

Weak Selectivity Predicted for Modeled Bundles of Viral Channel-Forming Protein E5 of Human Papillomavirus-16.

Protein E5 is a polytopic 83 amino acid membrane protein with three transmembrane domains (TMDs), encoded by high-risk human papillomavirus-16 (HPV-16). HPV-16 is found to be the causative agent for cervical cancer. Protein E5, among other proteins (e.g., E6, E7), is expressed at an "early" (E) stage when the cell turns malignant. It has been experimentally found that E5 forms hexameric assemblies, which show the characteristics of the class of so-called channel-forming proteins by rendering lipid membranes permeable to ions and small molecules. Protein E5 is used to achieve structural models of the protein in assembled bundles using a force field-based docking approach. Extended molecular dynamics simulations of selected bundles in fully hydrated lipid bilayers suggest the second TMD to be pore-lining, allowing for water columns to exist within the lumen of the pore. Full correlation analysis indicates asymmetric dynamics within the monomers of the bundle. Potential of mean force calculations of a snapshot structure of the putative open pore of the protein bundle propose low selectivity.

Viral channel proteins in intracellular protein-protein communication: Vpu of HIV-1, E5 of HPV16 and p7 of HCV.

Viral channel forming proteins are known for their capability to make the lipid membrane of the host cell and its subcellular compartments permeable to ions and small compounds. There is increasing evidence that some of the representatives of this class of proteins are also strongly interacting with host proteins and the effectiveness of this interaction seems to be high. Interaction of viral channel proteins with host factors has been proposed by bioinformatics approaches and has also been identified experimentally. An overview of the interactions with host proteins is given for Vpu from HIV-1, E5 from HPV-16 and p7 from HCV. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.