Unbinding and unfolding of adhesion protein complexes through stretching: Interplay between shear and tensile mechanical clamps

Using coarse‐grained molecular dynamics simulations, we analyze mechanically induced dissociation and unfolding of the protein complex CD48‐2B4. This heterodimer is an indispensable component of the immunological system: 2B4 is a receptor on natural killer cells whereas CD48 is expressed on surfaces of various immune cells. So far, its mechanostability has not been assessed either experimentally or theoretically. We find that the dissociation processes strongly depend on the direction of pulling and may take place in several pathways. Interestingly, the CD48‐2B4 interface can be divided into three distinct patches that act as units when resisting the pulling forces. At experimentally accessible pulling speeds, the characteristic mechanostability forces are in the range between 100 and 200 pN, depending on the pulling direction. These characteristic forces need not be associated with tensile forces involved in the act of separation of the complex because prior shear‐involving unraveling within individual proteins may give rise to a higher force peak. Proteins 2014; 82:3144–3153. © 2014 Wiley Periodicals, Inc.     

Formation of Cystine Slipknots in Dimeric Proteins

We consider mechanical stability of dimeric and monomeric proteins with the cystine knot motif. A structure based dynamical model is used to demonstrate that all dimeric and some monomeric proteins of this kind should have considerable resistance to stretching that is significantly larger than that of titin. The mechanisms of the large mechanostability are elucidated. In most cases, it originates from the induced formation of one or two cystine slipknots. Since there are four termini in a dimer, there are several ways of selecting two of them to pull by. We show that in the cystine knot systems, there is strong anisotropy in mechanostability and force patterns related to the selection. We show that the thermodynamic stability of the dimers is enhanced compared to the constituting monomers whereas machanostability is either lower or higher.     

Tuning the binding of coumarin 6 with DNA by molecular encapsulators: effect of β‐cyclodextrin and C‐hexylpyrogallol[4]arene

We report in this paper that the binding of coumarin 6 (C6) to DNA can be tuned by complexing it with host structures, viz. β‐cyclodextrin (β‐CD) and C‐hexylpyrogallol‐4‐arene (C‐HPA). Because host molecules are used as carriers of small molecules onto target sites, the exposed part of the guest molecule needs to be found out, and the relationship between the host : guest ratio and the mode of binding with the target macromolecule, that is, the DNA needs to be analyzed, in order to comprehend the preferred binding moiety and tune the binding. In this paper, the formation of the inclusion complex of C6 with β‐CD and with C‐HPA is studied by UV‐visible, fluorescence, 2D rotating‐frame nuclear Overhauser effect correlation spectroscopy and diffusion‐ordered spectroscopy nuclear magnetic resonance spectra and molecular modeling. C6 forms a 1:1 complex with β‐CD and a 1:2 complex with C‐HPA. The studies on the protonation of C6 in the presence and the absence of the host molecules suggest that the chromone part of C6 is outside the β‐CD molecule, whereas it is fully covered by C‐HPA. The binding of C6 with calf thymus DNA (ctDNA) occurs through intercalation and hydrogen bonding, and the host–guest structures remain intact on binding with ctDNA. The oxygens of the C6 molecules are exposed when inside the host molecules and aid in the hydrogen bonding with DNA. Copyright © 2014 John Wiley & Sons, Ltd.     

Integrating NMR,SAXS, and Atomistic Simulations: Structure and Dynamics of a Two-Domain Protein

Multidomain proteins with two or more independently folded functional domains are prevalent in nature. Whereas most multidomain proteins are linked linearly in sequence, roughly one-tenth possess domain insertions where a guest domain is implanted into a loop of a host domain, such that the two domains are connected by a pair of interdomain linkers. Here, we characterized the influence of the interdomain linkers on the structure and dynamics of a domain-insertion protein in which the guest LysM domain is inserted into a central loop of the host CVNH domain. Expanding upon our previous crystallographic and NMR studies, we applied SAXS in combination with NMR paramagnetic relaxation enhancement to construct a structural model of the overall two-domain system. Although the two domains have no fixed relative orientation, certain orientations were found to be preferred over others. We also assessed the accuracies of molecular mechanics force fields in modeling the structure and dynamics of tethered multidomain proteins by integrating our experimental results with microsecond-scale atomistic molecular dynamics simulations. In particular, our evaluation of two different combinations of the latest force fields and water models revealed that both combinations accurately reproduce certain structural and dynamical properties, but are inaccurate for others. Overall, our study illustrates the value of integrating experimental NMR and SAXS studies with long timescale atomistic simulations for characterizing structural ensembles of flexibly linked multidomain systems.     

Atomic force microscopy reveals a dual collagen‐binding activity for the staphylococcal surface protein SdrF

Staphylococcus epidermidis causes nosocomial infections by colonizing and forming biofilms on indwelling medical devices. This process involves specific interactions between cell wall‐anchored (CWA) proteins and host proteins adsorbed onto the biomaterial. Here, we have explored the molecular forces by which the S. epidermidis CWA protein serine‐aspartate repeat protein F (SdrF) binds to type I collagen, by means of advanced atomic force microscopy (AFM) techniques. Using single‐cell force spectroscopy, we found that SdrF mediates bacterial adhesion to collagen‐coated substrates through both weak and strong bonds. Single‐molecule force spectroscopy demonstrated that these bonds involve the A and B regions of SdrF, thus revealing that the protein is capable of dual ligand‐binding activity. Both weak and strong bonds showed high dissociation rates, indicating they are much less stable than those formed by the well‐characterized ‘dock, lock and latch’ mechanism. Collectively, our results show that CWA proteins can bind to ligands by novel mechanisms. We anticipate that AFM will greatly contribute to the identification of novel binding partners and binding mechanisms in staphylococcal CWA proteins.     

DNA‐doxorubicin interaction: New insights and peculiarities

We have investigated the interaction of the DNA molecule with the anticancer drug doxorubicin (doxo) by using three different experimental techniques: single molecule stretching, single molecule imaging, and dynamic light scattering. Such techniques allowed us to get new insights on the mechanical behavior of the DNA‐doxo complexes as well as on the physical chemistry of the interaction. First, the contour length data obtained from single molecule stretching were used to extract the physicochemical parameters of the DNA‐doxo interaction under different buffer conditions. This analysis has proven that the physical chemistry of such interaction can be modulated by changing the ionic strength of the surrounding buffer. In particular we have found that at low ionc strengths doxo interacts with DNA by simple intercalation (no aggregation) and/or by forming bound dimers. For high ionic strengths, otherwise, doxo‐doxo self‐association is enhanced, giving rise to the formation of bound doxo aggregates composed by 3 to 4 molecules along the double‐helix. On the other hand, the results obtained for the persistence length of the DNA‐doxo complexes is strongly force‐dependent, presenting different behaviors when measured with stretching or non‐stretching techniques.     

Mechanical Characterization of Protein L in the Low-Force Regime by Electromagnetic Tweezers/Evanescent Nanometry

Mechanical manipulation at the single molecule level of proteins exhibiting mechanical stability poses a technical challenge that has been almost exclusively approached by atomic force microscopy (AFM) techniques. However, due to mechanical drift limitations, AFM techniques are restricted to experimental recordings that last less than a minute in the high-force regime. Here we demonstrate a novel combination of electromagnetic tweezers and evanescent nanometry that readily captures the forced unfolding trajectories of protein L at pulling forces as low as 10 ∼ 15 pN. Using this approach, we monitor unfolding and refolding cycles of the same polyprotein for a period of time longer than 30 min. From such long-lasting recordings, we obtain ensemble averages of unfolding step sizes and rates that are consistent with single-molecule AFM data obtained at higher stretching forces. The unfolding kinetics of protein L at low stretching forces confirms and extends the observations that the mechanical unfolding rate is exponentially dependent on the pulling force within a wide range of stretching forces spanning from 13 pN up to 120 pN. Our experiments demonstrate a novel approach for the mechanical manipulation of single proteins for extended periods of time in the low-force regime.     

Preferred side‐chain conformation of arginine residues in a triple‐helical structure

The crystal structure of the triple‐helical peptide (Pro‐Hyp‐Gly)₃‐Pro‐Arg‐Gly‐(Pro‐Hyp‐Gly)₄ (POG3‐PRG‐POG4) was determined at 1.45 Å resolution. POG3‐PRG‐POG4 was designed to permit investigation of the side‐chain conformation of the Arg residues in a triple‐helical structure. Because of the alternative structure of one of three Arg residues, four side‐chain conformations were observed in an asymmetric unit. Among them, three adopt a ttg^?t conformation and the other adopts a tg^?g^?t conformation. A statistical analysis of 80 Arg residues in various triple‐helical peptides showed that, unlike those in globular proteins, they preferentially adopt a tt conformation for χ₁ and χ₂, as observed in POG3‐PRG‐POG4. This conformation permits van der Waals contacts between the side‐chain atoms of Arg and the main‐chain atoms of the adjacent strand in the same molecule. Unlike many other host–guest peptides, in which there is a significant difference between the helical twists in the guest and the host peptides, POG3‐PRG‐POG4 shows a marked difference between the helical twists in the N‐terminal peptide and those in the C‐terminal peptide, separated near the Arg residue. This suggested that the unique side‐chain conformation of the Arg residue affects not only the conformation of the guest peptide, but also the conformation of the peptide away from the Arg residue. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1000–1009, 2014.     

Predicting the order in which contacts are broken during single molecule protein stretching experiments

We combine two methods to enable the prediction of the order in which contacts are broken under external stretching forces in single molecule experiments. These two methods are Gō-like models and elastic network models. The Gō-like models have shown remarkable success in representing many aspects of protein behavior, including the reproduction of experimental data obtained from atomic force microscopy. The simple elastic network models are often used successfully to predict the fluctuations of residues around their mean positions, comparing favorably with the experimentally measured crystallographic B-factors. The behavior of biomolecules under external forces has been demonstrated to depend principally on their elastic properties and the overall shape of their structure. We have studied in detail the muscle protein titin and green fluorescent protein and tested for ten other proteins. First, we stretch the proteins computationally by performing stochastic dynamics simulations with the Gō-like model. We obtain the force-displacement curves and unfolding scenarios of possible mechanical unfolding. We then use the elastic network model to calculate temperature factors (B-factors) and compare the slowest modes of motion for the stretched proteins and compare them with the predicted order of breaking contacts between residues in the Gō-like model. Our results show that a simple Gaussian network model is able to predict contacts that break in the next time stage of stretching. Additionally, we have found that the contact disruption is strictly correlated with the highest force exerted by the backbone on these residues. Our prediction of bond-breaking agrees well with the unfolding scenario obtained with the Gō-like model. We anticipate that this method will be a useful new tool for interpreting stretching experiments.     

States and transitions during forced unfolding of a single spectrin repeat

Spectrin is a vital and abundant protein of the cytoskeleton. It has an elongated structure that is made by a chain of so-called spectrin repeats. Each repeat contains three antiparallel alpha-helices that form a coiled-coil structure. Spectrin forms an oligomeric structure that is able to cross-link actin filaments. In red cells, the spectrin/actin meshwork underlying cell membrane is thought to be responsible for special elastic properties of the cell. In order to determine mechanical unfolding properties of the spectrin repeat, we have used single molecule force spectroscopy to study the states of unfolding of an engineered polymeric protein consisting of identical spectrin domains. We demonstrate that the unfolding of spectrin domains can occur in a stepwise fashion during stretching. The force-extension patterns exhibit features that are compatible with the existence of at least one intermediate between the folded and the completely unfolded conformation. Only those polypeptides that still contain multiple intact repeats display intermediates, indicating a stabilisation effect. Precise force spectroscopy measurements on single molecules using engineered protein constructs reveal states and transitions during the mechanical unfolding of spectrin. Single molecule force spectroscopy appears to open a new window for the analysis of transition probabilities between different conformational states.     

IDENTIFYING COEVOLUTIONARY PATTERNS IN HUMAN LEUKOCYTE ANTIGEN (HLA) MOLECULES

The antigenic peptide, major histocompatibility complex molecule (MHC; also called human leukocyte antigen, HLA), coreceptor CD8, or CD4 and T‐cell receptor (TCR) function as a complex to initiate effectors’ mechanisms of the immune system. The tight functional and physical interaction among these molecules may have involved strong coevolution links among domains within and between proteins. Despite the importance of unraveling such dependencies to understand the arms race of host–pathogen interaction, no previous studies have aimed at achieving such an objective. Here, we perform an exhaustive coevolution analysis and show that indeed such dependencies are strongly shaping the evolution and probably the function of these molecules. We identify intramolecular coevolution in HLA class I and II at domains important for their immune activity. Most of the amino acid sites identified to be coevolving in HLAI have been also detected to undergo positive Darwinian selection highlighting therefore their adaptive value. We also identify coevolution among antigen‐binding pockets (P1‐P9) and among these and TCR‐binding sites. Conversely to HLAI, coevolution is weaker in HLAII. Our results support that such coevolutionary patterns are due to selective pressures of host–pathogen coevolution and cooperative binding of TCRs, antigenic peptides, and CD8/CD4 to HLAI and HLAII.     

Sop‐GPU: Accelerating biomolecular simulations in the centisecond timescale using graphics processors

Theoretical exploration of fundamental biological processes involving the forced unraveling of multimeric proteins, the sliding motion in protein fibers and the mechanical deformation of biomolecular assemblies under physiological force loads is challenging even for distributed computing systems. Using a C_α‐based coarse‐grained self organized polymer (SOP) model, we implemented the Langevin simulations of proteins on graphics processing units (SOP‐GPU program). We assessed the computational performance of an end‐to‐end application of the program, where all the steps of the algorithm are running on a GPU, by profiling the simulation time and memory usage for a number of test systems. The ～90‐fold computational speedup on a GPU, compared with an optimized central processing unit program, enabled us to follow the dynamics in the centisecond timescale, and to obtain the force‐extension profiles using experimental pulling speeds (v_f = 1–10 μm/s) employed in atomic force microscopy and in optical tweezers‐based dynamic force spectroscopy. We found that the mechanical molecular response critically depends on the conditions of force application and that the kinetics and pathways for unfolding change drastically even upon a modest 10‐fold increase in v_f. This implies that, to resolve accurately the free energy landscape and to relate the results of single‐molecule experiments in vitro and in silico, molecular simulations should be carried out under the experimentally relevant force loads. This can be accomplished in reasonable wall‐clock time for biomolecules of size as large as 10⁵ residues using the SOP‐GPU package. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Mapping HA‐tagged protein at the surface of living cells by atomic force microscopy

Single‐molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2‐adrenergic receptor (β2‐AR), a G protein‐coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2‐AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.     

Cytosolic Selection Systems To Study Protein Stability

Here we describe biosensors that provide readouts for protein stability in the cytosolic compartment of prokaryotes. These biosensors consist of tripartite sandwich fusions that link the in vitro stability or aggregation susceptibility of guest proteins to the in vivo resistance of host cells to the antibiotics kanamycin, spectinomycin, and nourseothricin. These selectable markers confer antibiotic resistance in a wide range of hosts and are easily quantifiable. We show that mutations within guest proteins that affect their stability alter the antibiotic resistances of the cells expressing the biosensors in a manner that is related to the in vitro stabilities of the mutant guest proteins. In addition, we find that polyglutamine tracts of increasing length are associated with an increased tendency to form amyloids in vivo and, in our sandwich fusion system, with decreased resistance to aminoglycoside antibiotics. We demonstrate that our approach allows the in vivo analysis of protein stability in the cytosolic compartment without the need for prior structural and functional knowledge.     

Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes

Plasmodium falciparum virulence is linked to its ability to sequester in post‐capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9‐96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence‐associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co‐exported with PfEMP1 into the host cell via vesicle‐like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface.     

Mechanical unfolding of covalently linked GroES: Evidence of structural subunit intermediates

It is difficult to determine the structural stability of the individual subunits or protomers of many proteins in the cell that exist in an oligomeric or complexed state. In this study, we used single‐molecule force spectroscopy on seven subunits of covalently linked cochaperonin GroES (ESC7) to evaluate the structural stability of the subunit. A modified form of ESC7 was immobilized on a mica surface. The force‐extension profile obtained from the mechanical unfolding of this ESC7 showed a distinctive sawtooth pattern that is typical for multimodular proteins. When analyzed according to the worm‐like chain model, the contour lengths calculated from the peaks in the profile suggested that linked‐GroES subunits unfold in distinct steps after the oligomeric ring structure of ESC7 is disrupted. The evidence that structured subunits of ESC7 withstand external force to some extent even after the perturbation of the oligomeric ring structure suggests that a stable monomeric intermediate is an important component of the equilibrium unfolding reaction of GroES.     

Piecewise All-Atom SMD Simulations Reveal Key Secondary Structures in Luciferase Unfolding Pathway

Although the folding of single-domain proteins is well characterized theoretically and experimentally, the folding of large multidomain proteins is less well known. Firefly luciferase, a 550 residue three-domain protein, has been commonly used as a substrate to study chaperone reactions and as a model system for the study of folding of long polypeptide chains, including related phenomena such as cotranslational folding. Despite being characterized by various experimental techniques, the atomic-level contributions of various secondary structures of luciferase to its fold’s mechanical stability remain unknown. Here, we developed a piecewise approach for all-atom steered molecular dynamics simulations to examine specific secondary structures that resist mechanical unfolding while minimizing the amount of computational resources required by the large water box of standard all-atom steered molecular dynamics simulations. We validated the robustness of this approach with a small NI3C protein and used our approach to elucidate the specific secondary structures that provide the largest contributions to luciferase mechanostability. In doing so, we show that piecewise all-atom steered molecular dynamics simulations can provide novel atomic resolution details regarding mechanostability and can serve as a platform for novel mutagenesis studies as well as a point for comparison with high-resolution force spectroscopy experiments.     

Principles and pitfalls in designing site-directed peptide ligands

An immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X-ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallo-graphically refined structures of 14 peptide–protein complexes at 2.7 Å resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change inconformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program anend-to-end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligandswere diffused into preexisting crystals. In sucha large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints. © 1993 Wiley-Liss, Inc.