Ying Fang

Flexibility is a mechanical determinant of antimicrobial activity for amphipathic cationic α-helical antimicrobial peptides.

Antimicrobial peptides (AMPs) are recognized as the potential substitutions for common antibiotics. Flexibility has been demonstrated to be a dominant on antimicrobial activity of an AMP, similar to the structural parameters such as hydrophobicity and hydrophobic moment as well as positive charge. To better understand the effect of flexibility on antimicrobial activity, we herein examined seventy-eight peptides derived from nine different species. Defined as a weighted average of amino acid flexibility indices over whole residue chain of AMP, flexibility index was used to scale the peptide flexibility and indicated to be a reflection of mechanical properties such as tensile and flexural rigidities. The results demonstrated that flexibility index is relevant to but different from other structural properties, may enhance activity against Escherichia coli for stiff clustered peptides or reduce activity against E. coli for flexible clustered peptides, and its optimum occurs at about -0.5. This effect of flexibility on antimicrobial activity may be involved to the antimicrobial actions, such as stable peptide-bound leaflet formation and sequent stress concentration in target cell membrane, mechanically. The present results provide a new insight in understanding antimicrobial actions and may be useful in seeking for a new structure-activity relationship for cationic and amphipathic α-helical peptides.

A rigidity-enhanced antimicrobial activity: a case for linear cationic α-helical peptide HP(2-20) and its four analogues.

Linear cationic α-helical antimicrobial peptides are referred to as one of the most likely substitutes for common antibiotics, due to their relatively simple structures (≤40 residues) and various antimicrobial activities against a wide range of pathogens. Of those, HP(2–20) was isolated from Helicobacter pylori ribosomal protein. To reveal a mechanical determinant that may mediate the antimicrobial activities, we examined the mechanical properties and structural stabilities of HP(2–20) and its four analogues of same chain length by steered molecular dynamics simulation. The results indicated the following: the resistance of H-bonds to the tensile extension mediated the early extensive stage; with the loss of H-bonds, the tensile force was dispensed to prompt the conformational phase transition; and Youngs moduli (N/m2) of the peptides were about 4∼8×109. These mechanical features were sensitive to the variation of the residue compositions. Furthermore, we found that the antimicrobial activity is rigidity-enhanced, that is, a harder peptide has stronger antimicrobial activity. It suggests that the molecular spring constant may be used to seek a new structure-activity relationship for different α-helical peptide groups. This exciting result was reasonably explained by a possible mechanical mechanism that regulates both the membrane pore formation and the peptide insertion.

Thermo-mechanical responses of a surface-coupled AFM cantilever

Atomic force microscopy (AFM) has been widely used for measuring mechanical properties of biological specimens such as cells, DNA, and proteins. This is usually done by monitoring deformations in response to controlled applied forces, which have to be at ultralow levels due to the extreme softness of the specimens. Consequently, such experiments may be susceptible to thermal excitations, manifested as force and displacement fluctuations that could reduce the measurement accuracy. To take advantage of, rather than to be limited by, such fluctuations, we have characterized the thermomechanical responses of an arbitrarily shaped AFM cantilever with the tip coupled to an elastic spring. Our analysis shows that the cantilever and the specimen behave as springs in parallel. This provides a method for determining the elasticity of the specimen by measuring the change in the tip fluctuations in the presence and absence of coupling. For rectangular and V-shaped cantilevers, we have derived a relationship between the mean-square deflection and the mean-square inclination and an approximate expression for the specimen spring constant in terms of contributions to the mean-square inclination from the first few vibration modes.

A Mechanism for Localized Dynamics-driven Affinity Regulation of the Binding of von Willebrand Factor to Platelet Glycoprotein Ibα

Background: Gain of function (GOF) mutations enhance the vWF-GPIbα interaction. Results: GOF mutations induce destabilization of the N-terminal arm and increase mobility of the α2-helix. Conclusion: Dynamics-driven up-regulation of A1 affinity to GPIbα serves as a GOF mechanism of type 2B mutations. Significance: These results are helpful in understanding the structural basis of GOF mutants and in developing allosteric drugs against the activated A1 domain. Binding of the A1 domain of von Willebrand factor (vWF) to glycoprotein Ibα (GPIbα) results in platelet adhesion, activation, and aggregation that initiates primary hemostasis. Both the elevated shear stress and the mutations associated with type 2B von Willebrand disease enhance the interaction between A1 and GPIbα. Through molecular dynamics simulations for wild-type vWF-A1 and its eight gain of function mutants (R543Q, I546V, ΔSS, etc.), we found that the gain of function mutations destabilize the N-terminal arm, increase a clock pendulum-like movement of the α2-helix, and turn a closed A1 conformation into a partially open one favoring binding to GPIbα. The residue Arg578 at the α2-helix behaves as a pivot in the destabilization of the N-terminal arm and a consequent dynamic change of the α2-helix. These results suggest a localized dynamics-driven affinity regulation mechanism for vWF-GPIbα interaction. Allosteric drugs controlling this intrinsic protein dynamics may be effective in blocking the GPIb-vWF interaction.

Force-dependent bond dissociation govern rolling of HL-60 cells through E-selectin.

E-selectin-mediated rolling on vascular surface of circulating leukocyte on vascular surface is a key initial event during inflammatory response and lymphocyte homing. This event depends not only on the specific interactions of adhesive molecules but also on the hemodynamics of blood flow. Little is still understood about whether wall shear stress or shear rate regulates the rolling. With flow chamber techniques, we here measured the effects of transport, shear stress and cell deformation on rolling of both unfixed and fixed HL-60 cells on E-selectin either in the absence or in the presence of 3% Ficoll in medium at various wall shear stresses from 0.05 to 0.7 dyn/cm(2). The results demonstrated a triphasic force-dependent rolling, that is, as increasing of force, the rolling would be accelerated firstly, then followed a decelerating phase occurred at the initial shear threshold of about 0.1 dyn/cm(2), and lastly returned to an accelerating process starting at the optimal shear threshold of 0.35 dyn/cm(2) approximately. The catch bond regime was completely reflected to rolling behaviors, such as tether lifetime, cell stop time and rolling velocity, meaning that the dominant factor to govern rolling is force. The initial shear threshold might be the minimum level of wall shear stress to sustain a stationary rolling, and the optimal shear threshold would make rolling to the most stable and regular. These findings strongly elucidate the catch bond mechanism for flow-enhanced rolling through E-selectin since longer bond lifetimes led to slower and stabler rolling.

Mapping Paratope on Antithrombotic Antibody 6B4 to Epitope on Platelet Glycoprotein Ibalpha via Molecular Dynamic Simulations

Binding of platelet receptor glycoprotein Ibα (GPIbα) to the A1 domain of von Willebrand factor (vWF) is a critical step in both physiologic hemostasis and pathologic thrombosis, for initiating platelet adhesion to subendothelium of blood vessels at sites of vascular injury. Gain-of-function mutations in GPIbα contribute to an abnormally high-affinity binding of platelets to vWF and can lead to thrombosis, an accurate complication causing heart attack and stroke. Of various antithrombotic monoclonal antibodies (mAbs) targeting human GPIbα, 6B4 is a potent one to inhibit the interaction between GPIbα and vWF-A1 under static and flow conditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route in researches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling, rigid body docking, free and steered molecular dynamics (MD) simulations, to identify key paratope residues on 6B4 and their partners on GPIbα, with hypothesis that the stable hydrogen bonds and salt bridges are the important linkers between paratope and epitope residues. Based on a best constructed model of 6B4 bound with GPIbα, the survival ratios and rupture times of all detected hydrogen bonds and salt bridges in binding site were examined via free and steered MD simulations and regarded as indices of thermal and mechanical stabilizations of the bonds, respectively. Five principal paratope residues with their partners were predicted with their high survival ratios and/or long rupture times of involved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top 5. Exciting, the present results were in good agreement with previous mutagenesis experiment data, meaning a wide application prospect of our novel computational procedure on researches of molecular of basis of ligand-receptor interactions, various antithrombotic mAbs and other antibodies as well as theoretically design of biomolecular drugs.

Bending rigidities of cell surface molecules P-selectin and PSGL-1

P-selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets. P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin expressed on leukocytes. The interaction of P-selectin and PSGL-1 mediates leukocyte tethering to and rolling on the vascular surface, which are initiating events in inflammatory and thrombotic processes. In the hemodynamic environment of the circulation, P-selectin and PSGL-1 are subject to a wide range of forces, which can cause deformation. For P-selectin/PSGL-1 interaction to be physically possible, these molecules may need to project above much of the glycocalyx layers of the respective cell surfaces, suggesting that they are either longer than the thickness of glycocalyx or better able to support compression than the glycocalyx. As such, the mechanical properties of these molecules and their functional implications merit investigation. Here we report determination of the bending rigidities of P-selectin and PSGL-1 by analyzing their thermally excited curvature fluctuations, whose values are of the order of magnitude of 100pNnm(2).

Flow-Enhanced Stability of Rolling Adhesion through E-Selectin

Selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on the vessel wall during inflammation. Extensive study has been devoted to elucidating the kinetic and mechanical constraints of receptor-ligand-interaction-mediated leukocyte adhesion, yet many questions remain unanswered. Here, we describe our design of an inverted flow chamber to compare adhesions of HL-60 cells to E-selectin in the upright and inverted orientations. This new, to our knowledge, design allowed us to evaluate the effect of gravity and to investigate the mechanisms of flow-enhanced adhesion. Cell rolling in the two orientations was qualitatively similar, and the quantitative differences can be explained by the effect of gravity, which promotes free-flowing cells to tether and detached cells to reattach to the surface in the upright orientation but prevents such attachment from happening in the inverted orientation. We characterized rolling stability by the lifetime of rolling adhesion and detachment of rolling cells, which could be easily measured in the inverted orientation, but not in the upright orientation because of the reattachment of transiently detached cells. Unlike the transient tether lifetime of E-selectin-ligand interaction, which exhibited triphasic slip-catch-slip bonds, the lifetime of rolling adhesion displayed a biphasic trend that first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. We have developed a minimal mathematical model for the probability of rolling adhesion. Comparison of the theoretical predictions to data has provided model validation and allowed evaluation of the effective two-dimensional association on-rate, kon, and the binding affinity, Ka, of the E-selectin-ligand interaction. kon increased with the wall shear stress from 0.1 to 0.7 dyn/cm(2). Ka firstxa0increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. Our results provide insights into how the interplay between flow-dependent on-rate and off-rate of E-selectin-ligand bonds determine flow-enhanced cell rolling stability.

A New Structure-Activity Relationship of Linear Cationic α-helical Antimicrobial Peptides

With the growing of pathogenic organisms’ resistibility to conventional antibiotics, antimicrobial peptides (AMPs), which have been isolated and characterized from tissues and organisms ranging from prokaryotes to humans, are recognized as a possible source of pharmaceuticals for the treatment of antibiotic-resistant bacterial infections. Many efforts have been made to increase the potency and specificity of these peptides so that they are toxic to microbes and not to mammals. A new structure-activity relationship of AMPs is revealed by analyzing the data of the antimicrobial assays here. Our results show that, not as in the receptor-mediated process, there is a linear correlation between the flexibility and the antimicrobial activity. The smaller the flexibility of the AMPs, the stronger the antimicrobial activity of the AMPs. This new structure-activity relationship may be used not only to predict the antimicrobial activity of AMPs from their sequences, but also in structural prediction, drug design and other AMPs’ researches.

Force-dependent calcium signaling and its pathway of human neutrophils on P-selectin in flow.

P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM), the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellular calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase (Syk). These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectin-induced calcium signaling of neutrophils in flow.