Impact of an alpha helix and a cysteine-cysteine disulfide bond on the resistance of bacterial adhesion pili to stress.

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that class 1a pili of uropathogenic E. coli (UPEC), as well as class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC class 5 pili. Examining structural and steered molecular dynamics simulation data, we find that this difference in class 1 pili subunit behavior originates from an α-helical motif that can unfold when exposed to force. A disulfide bond cross-linking ß-strands in class 1 pili stabilizes subunits, allowing them to tolerate higher forces than class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche, since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of class 5 ETEC pili.

Unveiling molecular interactions that stabilize bacterial adhesion pili.

Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers, we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal "staple" region of P pili, which provides interactions with pilins that are four and five subunits away, significantly stabilizes the helical filament structure. These data are consistent with previous structural data, and suggest that more layer-to-layer interactions could compensate for the lack of a staple in type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the function of pili is critically dependent on their structure and biophysical properties.

Long-ranged heterogeneous structure in aqueous solutions of the deep eutectic solvent choline and geranate at the liquid-vapor interface.

The deep eutectic solvent choline and geranate (CAGE) has shown promise in many therapeutic applications. CAGE facilitates drug delivery through unique modes of action making it an exciting therapeutic option. We examine the behavior of aqueous CAGE solutions at a liquid-vapor interface. We find that the liquid-vapor interface induces large oscillations in the density, which corresponds to spontaneous segregation into regions enriched with geranate and geranic acid and other regions enriched with water and choline. These heterogeneities are observed to extend nanometers into the liquid. Additionally, we find that the geranate and geranic acid orient so that their polar carboxyl or carboxylate groups are on average pointed toward the layer containing water and choline. Finally, we report surface tension and thermal expansion coefficients for various concentrations of aqueous CAGE. We find a non-monotonic trend in the surface tension with concentration. The structural and thermodynamic properties we report provide a new perspective on CAGE behavior, which helps deduce the action of CAGE in more sophisticated systems and inspire other studies and applications of CAGE and related materials.

The ionic liquid [C4mpy][Tf2N] induces bound-like structure in the intrinsically disordered protein FlgM.

The A. aeolicus intrinsically disordered protein FlgM has four well-defined α-helices when bound to σ28, but in water FlgM undergoes a change in tertiary structure. In this work, we investigate the structure of FlgM in aqueous solutions of the ionic liquid [C4mpy][Tf2N]. We find that FlgM is induced to fold by the addition of the ionic liquid, achieving average α-helicity values similar to the bound state. Analysis of secondary structure reveals significant similarity with the bound state, but the tertiary structure is found to be more compact. Interestingly, the ionic liquid is not homogeneously dispersed in the water, but instead aggregates near the protein. Separate simulations of aqueous ionic liquid do not show ion clustering, which suggests that FlgM stabilizes ionic liquid aggregation.

Steered molecular dynamics simulations of a type IV pilus probe initial stages of a force-induced conformational transition.

Type IV pili are long, protein filaments built from a repeating subunit that protrudes from the surface of a wide variety of infectious bacteria. They are implicated in a vast array of functions, ranging from bacterial motility to microcolony formation to infection. One of the most well-studied type IV filaments is the gonococcal type IV pilus (GC-T4P) from Neisseria gonorrhoeae, the causative agent of gonorrhea. Cryo-electron microscopy has been used to construct a model of this filament, offering insights into the structure of type IV pili. In addition, experiments have demonstrated that GC-T4P can withstand very large tension forces, and transition to a force-induced conformation. However, the details of force-generation, and the atomic-level characteristics of the force-induced conformation, are unknown. Here, steered molecular dynamics (SMD) simulation was used to exert a force in silico on an 18 subunit segment of GC-T4P to address questions regarding the nature of the interactions that lead to the extraordinary strength of bacterial pili. SMD simulations revealed that the buried pilin α1 domains maintain hydrophobic contacts with one another within the core of the filament, leading to GC-T4P's structural stability. At the filament surface, gaps between pilin globular head domains in both the native and pulled states provide water accessible routes between the external environment and the interior of the filament, allowing water to access the pilin α1 domains as reported for VC-T4P in deuterium exchange experiments. Results were also compared to the experimentally observed force-induced conformation. In particular, an exposed amino acid sequence in the experimentally stretched filament was also found to become exposed during the SMD simulations, suggesting that initial stages of the force induced transition are well captured. Furthermore, a second sequence was shown to be initially hidden in the native filament and became exposed upon stretching.

Effects of ATP and actin-filament binding on the dynamics of the myosin II S1 domain.

Actin and myosin interact with one another to perform a variety of cellular functions. Central to understanding the processive motion of myosin on actin is the characterization of the individual states along the mechanochemical cycle. We present an all-atom molecular dynamics simulation of the myosin II S1 domain in the rigor state interacting with an actin filament. We also study actin-free myosin in both rigor and post-rigor conformations. Using all-atom level and coarse-grained analysis methods, we investigate the effects of myosin binding on actin, and of actin binding on myosin. In particular, we determine the domains of actin and myosin that interact strongly with one another at the actomyosin interface using a highly coarse-grained level of resolution, and we identify a number of salt bridges and hydrogen bonds at the interface of myosin and actin. Applying coarse-grained analysis, we identify differences in myosin states dependent on actin-binding, or ATP binding. Our simulations also indicate that the actin propeller twist-angle and nucleotide cleft-angles are influenced by myosin at the actomyosin interface. The torsional rigidity of the myosin-bound filament is also calculated, and is found to be increased compared to previous simulations of the free filament.

Three structural solutions for bacterial adhesion pilus stability and superelasticity.

Bacterial adhesion pili are key virulence factors that mediate host-pathogen interactions in diverse epithelial environments. Deploying a multimodal approach, we probed the structural basis underpinning the biophysical properties of pili originating from enterotoxigenic (ETEC) and uropathogenic bacteria. Using cryo-electron microscopy we solved the structures of three vaccine target pili from ETEC bacteria, CFA/I, CS17, and CS20. Pairing these and previous pilus structures with force spectroscopy and steered molecular dynamics simulations, we find a strong correlation between subunit-subunit interaction energies and the force required for pilus unwinding, irrespective of genetic similarity. Pili integrate three structural solutions for stabilizing their assemblies: layer-to-layer interactions, N-terminal interactions to distant subunits, and extended loop interactions from adjacent subunits. Tuning of these structural solutions alters the biophysical properties of pili and promotes the superelastic behavior that is essential for sustained bacterial attachment.

The F-pilus biomechanical adaptability accelerates conjugative dissemination of antimicrobial resistance and biofilm formation.

Conjugation is used by bacteria to propagate antimicrobial resistance (AMR) in the environment. Central to this process are widespread conjugative F-pili that establish the connection between donor and recipient cells, thereby facilitating the spread of IncF plasmids among enteropathogenic bacteria. Here, we show that the F-pilus is highly flexible but robust at the same time, properties that increase its resistance to thermochemical and mechanical stresses. By a combination of biophysical and molecular dynamics methods, we establish that the presence of phosphatidylglycerol molecules in the F-pilus contributes to the structural stability of the polymer. Moreover, this structural stability is important for successful delivery of DNA during conjugation and facilitates rapid formation of biofilms in harsh environmental conditions. Thus, our work highlights the importance of F-pilus structural adaptations for the efficient spread of AMR genes in a bacterial population and for the formation of biofilms that protect against the action of antibiotics.

Assessing the Stability of Biological Fibrils by Molecular-Scale Simulations.

The nanomechanical characterization of several biological fibrils that are the result of protein aggregation via molecular dynamics simulation is nowadays feasible, and together with atomic force microscopy experiments has widened our understanding of the forces in the regime of pN-nN and system sizes of about hundreds of nanometers. Several methodologies have been developed to achieve this target, and they range from the atomistic representation via molecular force fields to coarse-grained strategies that provide comparable results with experiments in a systematic way. In this chapter, we discuss several methodologies for the calculation of mechanical parameters, such as the elastic constants of relevant biological systems. They are presented together with details about parameterization and current limitations. Then, we discuss some of the applications of such methodologies for the description of bacterial filament and ß-amyloid systems. Finally, the latest lines of development are discussed.

Quantitative determination of mechanical stability in the novel coronavirus spike protein.

We report on the novel observation about the gain in nanomechanical stability of the SARS-CoV-2 (CoV2) spike (S) protein in comparison with SARS-CoV from 2002 (CoV1). Our findings have several biological implications in the subfamily of coronaviruses, as they suggest that the receptor binding domain (RBD) (∼200 amino acids) plays a fundamental role as a damping element of the massive viral particle's motion prior to cell-recognition, while also facilitating viral attachment, fusion and entry. The mechanical stability via pulling of the RBD is 250 pN and 200 pN for CoV2 and CoV1 respectively, and the additional stability observed for CoV2 (∼50 pN) might play a role in the increasing spread of COVID-19.

Characterization of Structural and Energetic Differences between Conformations of the SARS-CoV-2 Spike Protein.

The novel coronavirus disease 2019 (COVID-19) pandemic has disrupted modern societies and their economies. The resurgence in COVID-19 cases as part of the second wave is observed across Europe and the Americas. The scientific response has enabled a complete structural characterization of the Severe Acute Respiratory Syndrome-novel Coronavirus 2 (SARS-CoV-2). Among the most relevant proteins required by the novel coronavirus to facilitate the cell entry mechanism is the spike protein. This protein possesses a receptor-binding domain (RBD) that binds the cellular angiotensin-converting enzyme 2 (ACE2) and then triggers the fusion of viral and host cell membranes. In this regard, a comprehensive characterization of the structural stability of the spike protein is a crucial step to find new therapeutics to interrupt the process of recognition. On the other hand, it has been suggested that the participation of more than one RBD is a possible mechanism to enhance cell entry. Here, we discuss the protein structural stability based on the computational determination of the dynamic contact map and the energetic difference of the spike protein conformations via the mapping of the hydration free energy by the Poisson-Boltzmann method. We expect our result to foster the discussion of the number of RBD involved during recognition and the repurposing of new drugs to disable the recognition by discovering new hotspots for drug targets apart from the flexible loop in the RBD that binds the ACE2.

Scope and efficacy of the broad-spectrum topical antiseptic choline geranate.

Choline geranate (also described as Choline And GEranic acid, or CAGE) has been developed as a novel biocompatible antiseptic material capable of penetrating skin and aiding the transdermal delivery of co-administered antibiotics. The antibacterial properties of CAGE were analyzed against 24 and 72 hour old biofilms of 11 clinically isolated ESKAPE pathogens (defined as Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter sp, respectively), including multidrug resistant (MDR) isolates. CAGE was observed to eradicate in vitro biofilms at concentrations as low as 3.56 mM (0.156% v:v) in as little as 2 hours, which represents both an improved potency and rate of biofilm eradication relative to that reported for most common standard-of-care topical antiseptics in current use. In vitro time-kill studies on 24 hour old Staphylococcus aureus biofilms indicate that CAGE exerts its antibacterial effect upon contact and a 0.1% v:v solution reduced biofilm viability by over three orders of magnitude (a 3log10 reduction) in 15 minutes. Furthermore, disruption of the protective layer of exopolymeric substances in mature biofilms of Staphylococcus aureus by CAGE (0.1% v:v) was observed in 120 minutes. Insight into the mechanism of action of CAGE was provided with molecular modeling studies alongside in vitro antibiofilm assays. The geranate ion and geranic acid components of CAGE are predicted to act in concert to integrate into bacterial membranes, affect membrane thinning and perturb membrane homeostasis. Taken together, our results show that CAGE demonstrates all properties required of an effective topical antiseptic and the data also provides insight into how its observed antibiofilm properties may manifest.

Molecular simulations of lactose-bound and unbound forms of the FaeG adhesin reveal critical amino acids involved in sugar binding.

F4 fimbriae are protein filaments found in enterotoxigenic Escherichia coli cells and are implicated in the process of bacterial infection due to their function as bacterial adhesins. These filaments are comprised from several proteins, but the bacterial adhesin FaeG, which is a lactose-binding protein, is the major subunit comprising F4 fimbriae. Crystal structures for three variants of the FaeG protein were recently solved, including the ad variant of FaeG that was crystallized in complex with lactose. However, the dynamics of the FaeG protein bound to lactose have not been explored previously using molecular dynamics simulations. Therefore, in order to study the dynamical interactions between the FaeG ad variant and lactose, we have carried out the first all-atom molecular dynamics simulations of this system. We have also probed the role of crystallographic water molecules on the stability of lactose in the FaeG binding site, and have simulated seven FaeG mutants to probe the influence of amino acid substitutions on the ability of FaeG to bind lactose effectively. Our simulations agree well with experimental results for the influence of mutations on lactose binding, provide dynamical insights into the interactions of FaeG with lactose, and also suggest the possibility of additional regions of the FaeG protein that may act as secondary lactose binding sites.

Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits.

Ions regulate the assembly and mechanical properties of actin filaments. Recent work using structural bioinformatics and site-specific mutagenesis favors the existence of two discrete and specific divalent cation binding sites on actin filaments, positioned in the long axis between actin subunits. Cation binding at one site drives polymerization, while the other modulates filament stiffness and plays a role in filament severing by the regulatory protein, cofilin. Existing structural methods have not been able to resolve filament-associated cations, and so in this work we turn to molecular dynamics simulations to suggest a candidate binding pocket geometry for each site and to elucidate the mechanism by which occupancy of the "stiffness site" affects filament mechanical properties. Incorporating a magnesium ion in the "polymerization site" does not seem to require any large-scale change to an actin subunit's conformation. Binding of a magnesium ion in the "stiffness site" adheres the actin DNase-binding loop (D-loop) to its long-axis neighbor, which increases the filament torsional stiffness and bending persistence length. Our analysis shows that bound D-loops occupy a smaller region of accessible conformational space. Cation occupancy buries key conserved residues of the D-loop, restricting accessibility to regulatory proteins and enzymes that target these amino acids.

Influence of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage.

We examine the effect of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage and contrast these results with the behavior of Trp-cage in water. We find the ionic liquid has a dramatic effect on Trp-cage, though many similarities with aqueous Trp-cage are observed. We assess Trp-cage folding by monitoring root mean square deviation from the crystallographic structure, radius of gyration, proline cis/trans isomerization state, protein secondary structure, amino acid contact formation and distance, and native and non-native contact formation. Starting from an unfolded configuration, Trp-cage folds in water at 298 K in less than 500 ns of simulation, but has very little mobility in the ionic liquid at the same temperature, which can be ascribed to the higher ionic liquid viscosity. At 365 K, the mobility of the ionic liquid is increased and initial stages of Trp-cage folding are observed, however Trp-cage does not reach the native folded state in 2 µs of simulation in the ionic liquid. Therefore, in addition to conventional molecular dynamics, we also employ scaled molecular dynamics to expedite sampling, and we demonstrate that Trp-cage in the ionic liquid does closely approach the aqueous folded state. Interestingly, while the reduced mobility of the ionic liquid is found to restrict Trp-cage motion, the ionic liquid does facilitate proline cis/trans isomerization events that are not seen in our aqueous simulations.

Electrostatic interactions between the Bni1p Formin FH2 domain and actin influence actin filament nucleation.

Formins catalyze nucleation and growth of actin filaments. Here, we study the structure and interactions of actin with the FH2 domain of budding yeast formin Bni1p. We built an all-atom model of the formin dimer on an Oda actin filament 7-mer and studied structural relaxation and interprotein interactions by molecular dynamics simulations. These simulations produced a refined model for the FH2 dimer associated with the barbed end of the filament and showed electrostatic interactions between the formin knob and actin target-binding cleft. Mutations of two formin residues contributing to these interactions (R1423N, K1467L, or both) reduced the interaction energies between the proteins, and in coarse-grained simulations, the formin lost more interprotein contacts with an actin dimer than with an actin 7-mer. Biochemical experiments confirmed a strong influence of these mutations on Bni1p-mediated actin filament nucleation, but not elongation, suggesting that different interactions contribute to these two functions of formins.

Electroluminescent zinc(II) bis(8-hydroxyquinoline): structural effects on electronic states and device performance.

We present direct evidence for stable oligomers in vacuum-deposited thin films of zinc(II) bis(8-hydroxyquinoline) (Znq(2)). The tetramer [(Znq(2))(4)] is the energetically favored configuration in both the single crystal and the vacuum-deposited thin film. Oligomerization leads to distinct, symmetry-driven differences between the electronic states in Znq(2) and those in the archetypal organic electroluminescent molecule tris(8-hydroxyquinoline) aluminum (Alq(3)). In the case of the Znq(2) tetramer, symmetry leads to an extended network of overlapping pyridyl and phenolato moieties in the solid film. Analysis of the electronic structure of (Znq(2))(4) calculated by ab initio Hartree-Fock (HF) methods reveals a localization and energy shift of high-lying occupied and low-lying unoccupied states on symmetry related ligands located on opposite sides of the supramolecular structure resulting in a dipole moment for (Znq(2))(4) tetramer close to zero. The optimal pi-overlap pathways, altered charge distributions, and extended electronic states of tetrameric Znq(2) may be expected to enable low operating voltage organic light-emitting devices (OLEDs) based on Znq(2). We present preliminary evidence that the operating voltage of (Znq(2))(4)-based OLEDs is indeed lower than that of identical devices made with Alq(3). Strategic substitution of 8-hydroxyquinoline ligands and control of the structural symmetry of the corresponding metal chelates may offer a route to high efficiency and low operating voltage small molecule OLEDs.