Ligand-dependent folding and unfolding dynamics and free energy landscapes of acylphosphatase.

Acylphosphatase (AcP) is an enzyme which catalyses the hydrolysis of acylphosphate. The binding with the phosphate ion (Pi) assumes significance in preserving both the stability and enzymatic activity of AcP. While previous studies using single molecule force spectroscopy explored the mechanical properties of AcP, the influence of Pi on its folding and unfolding dynamic behaviors remains unexplored. In this work, using stable magnetic tweezers, we measured and compared the force-dependent folding and unfolding rates of AcP in the Tris buffer and phosphate buffer within a force range from 2 pN to 40 pN. We found that Pi exerts no discernible effect on the folding dynamics but consistently decreases the force-dependent unfolding rate of AcP by a constant ratio across the entire force spectrum. The free energy landscapes of AcP in the absence and presence of Pi are constructed. Our results reveal that Pi selectively binds to the native state of AcP, stabilizing it and suggesting the general properties of specific ligand-receptor interactions.

Free Energy Landscape of Type III Fibronectin Domain with Identified Intermediate State and Hierarchical Symmetry.

The tenth domain of type III fibronectin (FNIII_{10}) mediates cell adhesion to the extracellular matrix. Despite its structural similarity to immunoglobulin domains, FNIII_{10} exhibits unique unfolding behaviors. We employed magnetic tweezers to investigate the unfolding and folding dynamics of FNIII_{10} under physiological forces (4-50 pN). Our results showed that FNIII_{10} follows a consistent transition pathway with an intermediate state characterized by detached A and G ß strands. We determined the folding free energies and all force-dependent transition rates of FNIII_{10} and found that both unfolding rates from the native state to the intermediate state and from the intermediate state to the unfolded state deviate from Bell's model. We constructed a quantitative free energy landscape with well-defined traps and barriers that exhibits a hierarchical symmetrical pattern. Our findings provide a comprehensive understanding of FNIII_{10} conformational dynamics and demonstrate how free energy landscape of multistate biomolecules can be precisely mapped, illuminating the relationship between thermal stability, intermediate states, and folding rates in protein folding.

Mechanical regulation of formin-dependent actin polymerization.

The actomyosin cytoskeleton network plays a key role in a variety of fundamental cellular processes such as cell division, migration, and cell adhesion. The functions of cytoskeleton rely on its capability to receive, generate, respond to and transmit mechanical signals throughout the cytoskeleton network within the cells and throughout the tissue via cell-extracellular matrix and cell-cell adhesions. Crucial to the cytoskeleton's functions is actin polymerization that is regulated by many cellular factors. Among these factors, the formin family proteins, which bind the barbed end of an actin filament (F-actin), are known to be a major actin polymerization promoting factor. Mounting evidence from single-molecule mechanical manipulation experiments have suggested that formin-dependent actin polymerization is sensitively regulated by the force and torque applied to the F-actin, making the formin family an emerging mechanosensing factor that selectively promotes elongation of the F-actin under tensile forces. In this review, we will focus on the current understanding of the mechanical regulation of formin-mediated actin polymerization, the key technologies that have enabled quantification of formin-mediated actin polymerization under mechanical constraints, and future perspectives and studies on molecular mechanisms involved in the mechanosensing of actin dynamics.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Mechanical Stabilization of a Bacterial Adhesion Complex.

The adhesions between Gram-positive bacteria and their hosts are exposed to varying magnitudes of tensile forces. Here, using an ultrastable magnetic tweezer-based single-molecule approach, we show the catch-bond kinetics of the prototypical adhesion complex of SD-repeat protein G (SdrG) to a peptide from fibrinogen ß (Fgß) over a physiologically important force range from piconewton (pN) to tens of pN, which was not technologically accessible to previous studies. At 37 °C, the lifetime of the complex exponentially increases from seconds at several pN to ∼1000 s as the force reaches 30 pN, leading to mechanical stabilization of the adhesion. The dissociation transition pathway is determined as the unbinding of a critical ß-strand peptide ("latch" strand of SdrG that secures the entire adhesion complex) away from its binding cleft, leading to the dissociation of the Fgß ligand. Similar mechanical stabilization behavior is also observed in several homologous adhesions, suggesting the generality of catch-bond kinetics in such bacterial adhesions. We reason that such mechanical stabilization confers multiple advantages in the pathogenesis and adaptation of bacteria.

Force-Dependent Interactions between Talin and Full-Length Vinculin.

Talin and vinculin are part of a multicomponent system involved in mechanosensing in cell-matrix adhesions. Both exist in autoinhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talin's interaction with vinculin has not been investigated. Here by quantifying the kinetics of force-dependent talin-vinculin interactions using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin, resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an autoinhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

Direct quantification of the translocation activities of Saccharomyces cerevisiae Pif1 helicase.

Saccharomyces cerevisiae Pif1 (ScPif1) is known as an ATP-dependent DNA helicase that plays critical roles in a number of important biological processes such as DNA replication, telomere maintenance and genome stability maintenance. Besides its DNA helicase activity, ScPif1 is also known as a single-stranded DNA (ssDNA) translocase, while how ScPif1 translocates on ssDNA is unclear. Here, by measuring the translocation activity of individual ScPif1 molecules on ssDNA extended by mechanical force, we identified two distinct types of ssDNA translocation. In one type, ScPif1 moves along the ssDNA track with a rate of ∼140 nt/s in 100 µM ATP, whereas in the other type, ScPif1 is immobilized to a fixed location of ssDNA and generates ssDNA loops against force. Between the two, the mobile translocation is the major form at nanomolar ScPif1 concentrations although patrolling becomes more frequent at micromolar concentrations. Together, our results suggest that ScPif1 translocates on extended ssDNA in two distinct modes, primarily in a 'mobile' manner.

Label-free Single-Molecule Quantification of Rapamycin-induced FKBP-FRB Dimerization for Direct Control of Cellular Mechanotransduction.

Chemically induced dimerization (CID) has been applied to study numerous biological processes and has important pharmacological applications. However, the complex multistep interactions under various physical constraints involved in CID impose a great challenge for the quantification of the interactions. Furthermore, the mechanical stability of the ternary complexes has not been characterized; hence, their potential application in mechanotransduction studies remains unclear. Here, we report a single-molecule detector that can accurately quantify almost all key interactions involved in CID and the mechanical stability of the ternary complex, in a label-free manner. Its application is demonstrated using rapamycin-induced heterodimerization of FRB and FKBP as an example. We revealed the sufficient mechanical stability of the FKBP/rapamycin/FRB ternary complex and demonstrated its utility in the precise switching of talin-mediated force transmission in integrin-based cell adhesions.

Force-Dependent Regulation of Talin-KANK1 Complex at Focal Adhesions.

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions.

Mechanical responses of the mechanosensitive unstructured domains in cardiac titin.

BACKGROUND INFORMATION: Titin is one of the three main filaments in cardiac sarcomere. Besides a chain of Ig domains, cardiac titin also contains a proline (P), glutamate (E), valine (V), lysine (K) (PEVK) domain and a cardiac-specific N2B domain, both are largely unstructured. While they are believed to be involved in the elastic (PEVK and N2B) and the trophic (N2B) functions of the heart, their mechanical responses in physiological level of forces remains poorly understood. RESULTS: In order to gain understanding on their mechanical responses, we used magnetic tweezers to investigate their force responses from 1 to 30 pN. We confirmed that in vitro the PEVK domain is intrinsically disordered within the force range. Surprisingly, we discovered a mechanosensitive folded element in the disordered region of N2B, ∼84 amino acids in length, which has a large folding energy of approximately -10 kB T. Based on the force responses of PEVK and N2B domains, as well as an approximated force-dependent unfolding and refolding rates of titin Ig domains, we show that the tension in cardiac titin fluctuates within 5 pN during cardiac contraction and extension cycle using Gillespie simulation algorithm. Exceptionally, the simulation shows that deletion of N2B domain results in 10-fold increase in peak force. CONCLUSION: Our results highlight a critical role that N2B may potentially play in regulating tension on cardiac titin. SIGNIFICANCE: The study provides new insights into the tension regulatory role of unstructured domains in the elastic function of the heart, which has broad implication in diastolic dysfunction and cardiac trophic mechanisms. In addition, the method can be applied to probing other unstructured mechanosensitive proteins/domains.

Bacillus subtilis RecA with DprA-SsbA antagonizes RecX function during natural transformation.

Bacillus subtilis DprA and RecX proteins, which interact with RecA, are crucial for efficient chromosomal and plasmid transformation. We showed that RecA, in the rATP·Mg2+ bound form (RecA·ATP), could not compete with RecX, SsbA or SsbB for assembly onto single-stranded (ss)DNA, but RecA·dATP partially displaced these proteins from ssDNA. RecX promoted reversible depolymerization of preformed RecA·ATP filaments. The two-component DprA-SsbA mediator reversed the RecX negative effect on RecA filament extension, but not DprA or DprA and SsbB. In the presence of DprA-SsbA, RecX added prior to RecA·ATP inhibited DNA strand exchange, but this inhibition was reversed when RecX was added after RecA. We propose that RecA nucleation is more sensitive to RecX action than is RecA filament growth. DprA-SsbA facilitates formation of an active RecA filament that directly antagonizes the inhibitory effects of RecX. RecX and DprA enable chromosomal transformation by altering RecA filament dynamics. DprA-SsbA and RecX proteins constitute a new regulatory network of RecA function. DprA-SsbA contributes to the formation of an active RecA filament and directly antagonizes the inhibitory effects of RecX during natural transformation.

Parallel triplex structure formed between stretched single-stranded DNA and homologous duplex DNA.

The interaction between the single-stranded DNA and the homologous duplex DNA is essential for DNA homologous repair. Here, we report that parallel triplex structure can form spontaneously between a mechanically extended ssDNA and a homologous dsDNA in protein-free condition. The triplex has a contour length close to that of a B-form DNA duplex and remains stable after force is released. The binding energy between the ssDNA and the homologous dsDNA in the triplex is estimated to be comparable to the basepairing energy in a B-form dsDNA. As ssDNA is in a similar extended conformation within recombinase-coated nucleoprotein filaments, we propose that the parallel triplex may form and serve as an intermediate during recombinase-catalyzed homologous joint formation.

Effects of Mechanical Stimuli on Profilin- and Formin-Mediated Actin Polymerization.

Self-assembling actin filaments not only form the basis of the cytoskeleton network in cells but also are utilized as nanosized building blocks to make novel active matter in which the dynamic polymerization and depolymerization of actin filaments play a key role. Formins belong to a main family of actin nucleation factors that bind to the barbed end of actin filaments and regulate actin polymerization through an interaction with profilin. Due to actomyosin contractility and relative rotation between formin and actin filaments, formin-dependent actin polymerization is subject to force and rotation constraints. However, it remains unclear how force and rotation constraints affect formin-dependent actin polymerization in the presence of profilin. Here, we show that for rotation-unconstrained actin filaments, elongation is accelerated by both force and profilin. The combined effect leads to surprisingly fast actin elongation that can approach the diffusion-limited rate at forces of a few piconewtons. The elongation of rotation-constrained filaments is also accelerated by profilin but is insensitive to applied force. We show that FH2, the main actin binding domain, plays the primary mechanosensing role. Together, the findings not only significantly advance our understanding of the mechanochemical regulation of formin-mediated actin polymerization in cells but also can potentially be utilized to make novel actin-based active matter.

Phosphorylation Reduces the Mechanical Stability of the α-Catenin/ ß-Catenin Complex.

The α-catenin/ß-catenin complex serves as a critical molecular interface involved in cadherin-catenin-based mechanosensing at the cell-cell adherence junction that plays a critical role in tissue integrity, repair, and embryonic development. This complex is subject to tensile forces due to internal actomyosin contractility and external mechanical micro-environmental perturbation. However, the mechanical stability of this complex has yet to be quantified. Here, we directly quantified the mechanical stability of the α-catenin/ß-catenin complex and showed that it has enough mechanical stability to survive for tens to hundreds of seconds within physiological level of forces up to 10 pN. Phosphorylation or phosphotyrosine-mimetic mutations (Y142E or/and T120E) on ß-catenin shorten the mechanical lifetime of the complex by tens of fold over the same force range. These results provide insights into the regulation of the α-catenin/ß-catenin complex by phosphorylation.

Uncovering mechanosensing mechanisms at the single protein level using magnetic tweezers.

Mechanosensing of the micro-environments has been shown to be essential for cell survival, growth, differentiation and migration. The mechanosensing pathways are mediated by a set of mechanosensitive proteins located at focal adhesion and cell-cell adherens junctions as well as in the cytoskeleton network. Here we review the applications of magnetic tweezers on elucidating the molecular mechanisms of the mechanosensing proteins. The scope of this review includes the principles of the magnetic tweezers technology, theoretical analysis of force-dependent stability and interaction of mechanosensing proteins, and recent findings obtained using magnetic tweezers.

Disturbance-free rapid solution exchange for magnetic tweezers single-molecule studies.

Single-molecule manipulation technologies have been extensively applied to studies of the structures and interactions of DNA and proteins. An important aspect of such studies is to obtain the dynamics of interactions; however the initial binding is often difficult to obtain due to large mechanical perturbation during solution introduction. Here, we report a simple disturbance-free rapid solution exchange method for magnetic tweezers single-molecule manipulation experiments, which is achieved by tethering the molecules inside microwells (typical dimensions-diameter (D): 40-50 µm, height (H): 100 µm; H:D∼2:1). Our simulations and experiments show that the flow speed can be reduced by several orders of magnitude near the bottom of the microwells from that in the flow chamber, effectively eliminating the flow disturbance to molecules tethered in the microwells. We demonstrate a wide scope of applications of this method by measuring the force dependent DNA structural transitions in response to solution condition change, and polymerization dynamics of RecA on ssDNA/SSB-coated ssDNA/dsDNA of various tether lengths under constant forces, as well as the dynamics of vinculin binding to α-catenin at a constant force (< 5 pN) applied to the α-catenin protein.

Elasticity of the Transition State Leading to an Unexpected Mechanical Stabilization of Titin Immunoglobulin Domains.

The giant protein titin plays a critical role in regulating the passive elasticity of muscles, mainly through the stochastic unfolding and refolding of its numerous immunoglobulin domains in the I-band of sarcomeres. The unfolding dynamics of titin immunoglobulin domains at a force range greater than 100 pN has been studied by atomic force microscopy, while that at smaller physiological forces has not been measured before. By using magnetic tweezers, it is found that the titin I27 domain unfolds in a surprising non-monotonic force-dependent manner at forces smaller than 100 pN, with the slowest unfolding rate occurring around 22 pN. We further demonstrate that a model with single unfolding pathway taking into account the elasticity of the transition state can reproduce the experimental results. These results provide important novel insights into the regulation mechanism of the passive elasticity of muscle tissues.

Mechanical force antagonizes the inhibitory effects of RecX on RecA filament formation in Mycobacterium tuberculosis.

Efficient bacterial recombinational DNA repair involves rapid cycles of RecA filament assembly and disassembly. The RecX protein plays a crucial inhibitory role in RecA filament formation and stability. As the broken ends of DNA are tethered during homologous search, RecA filaments assembled at the ends are likely subject to force. In this work, we investigated the interplay between RecX and force on RecA filament formation and stability. Using magnetic tweezers, at single molecular level, we found that Mycobacterium tuberculosis (Mt) RecX could catalyze stepwise de-polymerization of preformed MtRecA filament in the presence of ATP hydrolysis at low forces (<7 pN). However, applying larger forces antagonized the inhibitory effects of MtRecX, and a partially de-polymerized MtRecA filament could re-polymerize in the presence of MtRecX, which cannot be explained by previous models. Theoretical analysis of force-dependent conformational free energies of naked ssDNA and RecA nucleoprotein filament suggests that mechanical force stabilizes RecA filament, which provides a possible mechanism for the observation. As the antagonizing effect of force on the inhibitory function of RecX takes place in a physiological range; these findings broadly suggest a potential mechanosensitive regulation during homologous recombination.

Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry.

Double-stranded DNA (dsDNA) unconstrained by torsion undergoes an overstretching transition at about 65 pN, elongating the DNA to about 1.7-fold. Three possible structural transitions have been debated for the nature of DNA overstretching: (i) "peeling" apart of dsDNA to produce a peeled ssDNA strand under tension while the other strand coils, (ii) "inside-strand separation" of dsDNA to two parallel ssDNA strands that share tension (melting bubbles), and (iii) "B-to-S" transition to a novel dsDNA, termed S-DNA. Here we overstretched an end-opened DNA (with one open end to allow peeling) and an end-closed (i.e., both ends of the linear DNA are covalently closed to prohibit peeling) and torsion-unconstrained DNA. We report that all three structural transitions exist depending on experimental conditions. For the end-opened DNA, the peeling transition and the B-to-S transition were observed; for the end-closed DNA, the inside-strand separation and the B-to-S transition were observed. The peeling transition and the inside-strand separation are hysteretic and have an entropy change of approximately 17 cal/(K⋅mol), whereas the B-to-S transition is nonhysteretic and has an entropy change of approximately -2 cal/(K⋅mol). The force-extension curves of peeled ssDNA, melting bubbles, and S-DNA were characterized by experiments. Our results provide experimental evidence for the formation of DNA melting bubbles driven by high tension and prove the existence of nonmelted S-DNA. Our findings afford a full understanding of three possible force-driven structural transitions of torsion-unconstrained DNA and the resulting three overstretched DNA structures.

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein.

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.