Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.

Two conserved second-sphere ßArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase ßR52A, ßR52K, ßR157A, ßR157K and ReNHase ßR61A) were successfully expressed and purified. Apart from the PtNHase ßR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase ßR mutant enzymes were between 1.8 and 12.4 s-1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ∼10 to ∼40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase ßR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere ßR residues in the proposed subunit swapping process and post-translational modification of the α-subunit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.

A Perspective on Developing Modeling and Image Analysis Tools to Investigate Mechanosensing Proteins.

The shift of funding organizations to prioritize interdisciplinary work points to the need for workflow models that better accommodate interdisciplinary studies. Most scientists are trained in a specific field and are often unaware of the kind of insights that other disciplines could contribute to solving various problems. In this paper, we present a perspective on how we developed an experimental pipeline between a microscopy and image analysis/bioengineering lab. Specifically, we connected microscopy observations about a putative mechanosensing protein, obscurin, to image analysis techniques that quantify cell changes. While the individual methods used are well established (fluorescence microscopy; ImageJ WEKA and mTrack2 programs; MATLAB), there are no existing best practices for how to integrate these techniques into a cohesive, interdisciplinary narrative. Here, we describe a broadly applicable workflow of how microscopists can more easily quantify cell properties (e.g., perimeter, velocity) from microscopy videos of eukaryotic (MDCK) adherent cells. Additionally, we give examples of how these foundational measurements can create more complex, customizable cell mechanics tools and models.

The N-terminus of obscurin is flexible in solution.

The N-terminal half of the giant cytoskeletal protein obscurin is comprised of more than 50 Ig-like domains, arranged in tandem. Domains 18-51 are connected to each other through short 5-residue linkers, and this arrangement has been previously shown to form a semi-flexible rod in solution. Domains 1-18 generally have slightly longer ~7 residue interdomain linkers, and the multidomain structure and motion conferred by this kind of linker is understudied. Here, we use NMR, SAXS, and MD to show that these longer linkers are associated with significantly more domain/domain flexibility, with the resulting multidomain structure being moderately compact. Further examination of the relationship between interdomain flexibility and linker length shows there is a 5 residue "sweet spot" linker length that results in dual-domain systems being extended, and conversely that both longer or shorter linkers result in a less extended structure. This detailed knowledge of the obscurin N terminus structure and flexibility allowed for mathematical modeling of domains 1-18, which suggests that this region likely forms tangles if left alone in solution. Given how infrequently protein tangles occur in nature, and given the pathological outcomes that occur when tangles do arise, our data suggest that obscurin is likely either significantly scaffolded or else externally extended in the cell.

An integrated biophysical approach to discovering mechanisms of NDM-1 inhibition for several thiol-containing drugs.

Due to the rapid proliferation of antibiotic-resistant pathogenic bacteria, known as carbapenem-resistant enterobacteriaceae, the efficacy of ß-lactam antibiotics is threatened. ß-lactam antibiotics constitute over 50% of the available antibiotic arsenal. Recent efforts have been focused on developing inhibitors to these enzymes. In an effort to understand the mechanism of inhibition(s) of four FDA-approved thiol-containing drugs that were previously reported to be inhibitors of New Delhi metallo-ß-lactamase (NDM-1), various biochemical and spectroscopic techniques were used. Isothermal titration calorimetry demonstrated the binding affinity to NDM-1 corresponds to the reported IC50 values of the inhibitors. Equilibrium dialyses and metal analyses demonstrated that all of these inhibitors formed ternary complexes with ZnZn-NDM-1. Spectroscopic studies on CoCo-NDM-1 revealed two distinct binding modes for the thiol-containing compounds. These findings validate the need to further investigate the mechanism of inhibition of MBL inhibitors. Further research to identify inhibition capabilities beyond reported IC50 values is necessary for understanding the binding modes of these identified compounds and to provide the necessary foundation for developing clinically relevant MBL inhibitors.

A Single Salt Bridge in VIM-20 Increases Protein Stability and Antibiotic Resistance under Low-Zinc Conditions.

To understand the evolution of Verona integron-encoded metallo-ß-lactamase (VIM) genes (blaVIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-ß-lactamase families, although compensating mutations use different mechanisms to enhance resistance.IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-ß-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-ß-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.

Emergent spatiotemporal dynamics of the actomyosin network in the presence of chemical gradients.

We used particle-based computer simulations to study the emergent properties of the actomyosin cytoskeleton. Our model accounted for biophysical interactions between filamentous actin and non-muscle myosin II and was motivated by recent experiments demonstrating that spatial regulation of myosin activity is required for fibroblasts responding to spatial gradients of platelet derived growth factor (PDGF) to undergo chemotaxis. Our simulations revealed the spontaneous formation of actin asters, consistent with the punctate actin structures observed in chemotacting fibroblasts. We performed a systematic analysis of model parameters to identify biochemical steps in myosin activity that significantly affect aster formation and performed simulations in which model parameter values vary spatially to investigate how the model responds to chemical gradients. Interestingly, spatial variations in motor stiffness generated time-dependent behavior of the actomyosin network, in which actin asters continued to spontaneously form and dissociate in different regions of the gradient. Our results should serve as a guide for future experimental investigations.

Emergent mechanics of actomyosin drive punctuated contractions and shape network morphology in the cell cortex.

Filamentous actin (F-actin) and non-muscle myosin II motors drive cell motility and cell shape changes that guide large scale tissue movements during embryonic morphogenesis. To gain a better understanding of the role of actomyosin in vivo, we have developed a two-dimensional (2D) computational model to study emergent phenomena of dynamic unbranched actomyosin arrays in the cell cortex. These phenomena include actomyosin punctuated contractions, or "actin asters" that form within quiescent F-actin networks. Punctuated contractions involve both formation of high intensity aster-like structures and disassembly of those same structures. Our 2D model allows us to explore the kinematics of filament polarity sorting, segregation of motors, and morphology of F-actin arrays that emerge as the model structure and biophysical properties are varied. Our model demonstrates the complex, emergent feedback between filament reorganization and motor transport that generate as well as disassemble actin asters. Since intracellular actomyosin dynamics are thought to be controlled by localization of scaffold proteins that bind F-actin or their myosin motors we also apply our 2D model to recapitulate in vitro studies that have revealed complex patterns of actomyosin that assemble from patterning filaments and motor complexes with microcontact printing. Although we use a minimal representation of filament, motor, and cross-linker biophysics, our model establishes a framework for investigating the role of other actin binding proteins, how they might alter actomyosin dynamics, and makes predictions that can be tested experimentally within live cells as well as within in vitro models.

A Noncanonical Metal Center Drives the Activity of the Sediminispirochaeta smaragdinae Metallo-ß-lactamase SPS-1.

In an effort to evaluate whether a recently reported putative metallo-ß-lactamase (MßL) contains a novel MßL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not ß-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar to that in NDM-1 and other subclass B1 MßLs; however, the Zn1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 Å from the position reported in other MßLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of ß-lactam antibiotics. This study reveals a novel metal binding site in MßLs and suggests that the targeting of metal binding sites in MßLs with inhibitors is now more challenging with the identification of this new MßL.

Evolution of New Delhi metallo-ß-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used ß-lactamase inhibitors to counter the activity of metallo-ß-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-ß-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of ß-lactam antibiotics and zinc deprivation.

Clinical Variants of New Delhi Metallo-ß-Lactamase Are Evolving To Overcome Zinc Scarcity.

Use and misuse of antibiotics have driven the evolution of serine ß-lactamases to better recognize new generations of ß-lactam drugs, but the selective pressures driving evolution of metallo-ß-lactamases are less clear. Here, we present evidence that New Delhi metallo-ß-lactamase (NDM) is evolving to overcome the selective pressure of zinc(II) scarcity. Studies of NDM-1, NDM-4 (M154L), and NDM-12 (M154L, G222D) demonstrate that the point mutant M154L, contained in 50% of clinical NDM variants, selectively enhances resistance to the penam ampicillin at low zinc(II) concentrations relevant to infection sites. Each of the clinical variants is shown to be progressively more thermostable and to bind zinc(II) more tightly than NDM-1, but a selective enhancement of penam turnover at low zinc(II) concentrations indicates that most of the improvement derives from catalysis rather than stability. X-ray crystallography of NDM-4 and NDM-12, as well as bioinorganic spectroscopy of dizinc(II), zinc(II)/cobalt(II), and dicobalt(II) metalloforms probe the mechanism of enhanced resistance and reveal perturbations of the dinuclear metal cluster that underlie improved catalysis. These studies support the proposal that zinc(II) scarcity, rather than changes in antibiotic structure, is driving the evolution of new NDM variants in clinical settings.

Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-ß-lactamase-1.

The efficacy of ß-lactam antibiotics is threatened by the emergence and global spread of metallo-ß-lactamase (MBL) mediated resistance, specifically New Delhi metallo-ß-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related ß-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Actomyosin meshwork mechanosensing enables tissue shape to orient cell force.

Sculpting organism shape requires that cells produce forces with proper directionality. Thus, it is critical to understand how cells orient the cytoskeleton to produce forces that deform tissues. During Drosophila gastrulation, actomyosin contraction in ventral cells generates a long, narrow epithelial furrow, termed the ventral furrow, in which actomyosin fibres and tension are directed along the length of the furrow. Using a combination of genetic and mechanical perturbations that alter tissue shape, we demonstrate that geometrical and mechanical constraints act as cues to orient the cytoskeleton and tension during ventral furrow formation. We developed an in silico model of two-dimensional actomyosin meshwork contraction, demonstrating that actomyosin meshworks exhibit an inherent force orienting mechanism in response to mechanical constraints. Together, our in vivo and in silico data provide a framework for understanding how cells orient force generation, establishing a role for geometrical and mechanical patterning of force production in tissues.

Nano-Cesium for Anti-Cancer Properties: An Investigation into Cesium Induced Metabolic Interference.

The use of cesium chloride (CsCl) for cancer therapy ("high pH therapy") has been theorized to produce anticancer properties by raising intracellular pH to induce apoptosis. Although considered as "alternative medicine", little scientific evidence supports this theory. Alternatively, cells have no cesium ion (Cs+) mediated channels for clearance. Thus, such unstable electrochemical distributions have the severe potential to disrupt electrochemical dependent cellular processes, such as glucose cotransporters. Hence, a detailed investigation of pH changing effects and glucose uptake inhibition are warranted as a possible cesium-induced anticancer therapy. We developed and characterized cesium nanoparticles (38 ± 6 nm), termed NanoCs, for nanoparticle-mediated internalization of the ion, and compared its treatment to free CsCl. Our investigations suggest that neither NanoCs nor CsCl drastically changed the intracellular pH, negating the theory. Alternatively, NanoCs lead to a significant decrease in glucose uptake when compared to free CsCl, suggesting cesium inhibited glucose uptake. An apoptosis assay of observed cell death affirms that NanoCs leads tumor cells to initiate apoptosis rather than follow necrotic behavior. Furthermore, NanoCs lead to in vivo tumor regression, where H&E analysis confirmed apoptotic cell populations. Thus, NanoCs performed pH-independent anticancer therapy by inducing metabolic stasis.

Biomechanics and the thermotolerance of development.

Successful completion of development requires coordination of patterning events with morphogenetic movements. Environmental variability challenges this coordination. For example, developing organisms encounter varying environmental temperatures that can strongly influence developmental rates. We hypothesized that the mechanics of morphogenesis would have to be finely adjusted to allow for normal morphogenesis across a wide range of developmental rates. We formulated our hypothesis as a simple model incorporating time-dependent application of force to a viscoelastic tissue. This model suggested that the capacity to maintain normal morphogenesis across a range of temperatures would depend on how both tissue viscoelasticity and the forces that drive deformation vary with temperature. To test this model we investigated how the mechanical behavior of embryonic tissue (Xenopus laevis) changed with temperature; we used a combination of micropipette aspiration to measure viscoelasticity, electrically induced contractions to measure cellular force generation, and confocal microscopy to measure endogenous contractility. Contrary to expectations, the viscoelasticity of the tissues and peak contractile tension proved invariant with temperature even as rates of force generation and gastrulation movements varied three-fold. Furthermore, the relative rates of different gastrulation movements varied with temperature: the speed of blastopore closure increased more slowly with temperature than the speed of the dorsal-to-ventral progression of involution. The changes in the relative rates of different tissue movements can be explained by the viscoelastic deformation model given observed viscoelastic properties, but only if morphogenetic forces increase slowly rather than all at once.

The interplay between cell signalling and mechanics in developmental processes.

Force production and the propagation of stress and strain within embryos and organisms are crucial physical processes that direct morphogenesis. In addition, there is mounting evidence that biomechanical cues created by these processes guide cell behaviours and cell fates. In this Review we discuss key roles for biomechanics during development to directly shape tissues, to provide positional information for cell fate decisions and to enable robust programmes of development. Several recently identified molecular mechanisms suggest how cells and tissues might coordinate their responses to biomechanical cues. Finally, we outline long-term challenges in integrating biomechanics with genetic analysis of developing embryos.

Rotational model for actin filament alignment by myosin.

Dynamics of the actomyosin cytoskeleton regulate cellular processes such as secretion, cell division, cell motility, and shape change. Actomyosin dynamics are themselves regulated by proteins that control actin filament polymerization and depolymerization, and myosin motor contractility. Previous theoretical work has focused on translational movement of actin filaments but has not considered the role of filament rotation. Since filament rotational movements are likely sources of forces that direct cell shape change and movement we explicitly model the dynamics of actin filament rotation as myosin II motors traverse filament pairs, drawing them into alignment. Using Monte Carlo simulations we find an optimal motor velocity for alignment of actin filaments. In addition, when we introduce polymerization and depolymerization of actin filaments, we find that alignment is reduced and the filament arrays exist in a stable, asynchronous state. Further analysis with continuum models allows us to investigate factors contributing to the stability of filament arrays and their ability to generate force. Interestingly, we find that two different morphologies of F-actin arrays generate the same amount of force. We also identify a phase transition to alignment which occurs when either polymerization rates are reduced or motor velocities are optimized. We have extended our analysis to include a maximum allowed stretch of the myosin motors, and a non-uniform length for filaments leading to little change in the qualitative results. Through the integration of simulations and continuum analysis, we are able to approach the problem of understanding rotational alignment of actin filaments by myosin II motors.