Stiffness of Cargo–Motor Linkage Tunes Myosin VI Motility and Response to Load

Rachit Shrivastava, Ashim Rai, Murti Salapaka, Sivaraj Sivaramakrishnan

We examine the effect of cargo–motor linkage stiffness on the mechanobiological properties of the molecular motor myosin VI. We use the programmability of DNA nanostructures to modulate cargo–motor linkage stiffness and combine it with high-precision optical trapping measurements to measure the effect of linkage stiffness on the motile properties of myosin VI. Our results reveal that a stiff cargo–motor linkage leads to shorter step sizes and load-induced anchoring of myosin VI, while a flexible linkage results in longer steps with frequent detachments from the actin filament under load. Our findings suggest a novel regulatory mechanism for tuning the dual cellular roles of the anchor and transporter ascribed to myosin VI.

DOI

A Tour de Force on the Double Helix: Exploiting DNA Mechanics To Study DNA-Based Molecular Machines

Michael R. Wasserman and Shixin Liu

DNA is both a fundamental building block of life and a fascinating natural polymer. The advent of single-molecule manipulation tools made it possible to exert controlled force on individual DNA molecules and measure their mechanical response. Such investigations elucidated the elastic properties of DNA and revealed its distinctive structural configurations across force regimes. In the meantime, a detailed understanding of DNA mechanics laid the groundwork for single-molecule studies of DNA-binding proteins and DNA-processing enzymes that bend, stretch, and twist DNA. These studies shed new light on the metabolism and transactions of nucleic acids, which constitute a major part of the cell’s operating system. Furthermore, the marriage of single-molecule fluorescence visualization and force manipulation has enabled researchers to directly correlate the applied tension to changes in the DNA structure and the behavior of DNA-templated complexes. Overall, experimental exploitation of DNA mechanics has been and will continue to be a unique and powerful strategy for understanding how molecular machineries recognize and modify the physical state of DNA to accomplish their biological functions.

DOI

Stiffness of Cargo–Motor Linkage Tunes Myosin VI Motility and Response to Load

Rachit Shrivastava, Ashim Rai, Murti Salapaka Sivaraj Sivaramakrishnan
We examine the effect of cargo–motor linkage stiffness on the mechanobiological properties of the molecular motor myosin VI. We use the programmability of DNA nanostructures to modulate cargo–motor linkage stiffness and combine it with high-precision optical trapping measurements to measure the effect of linkage stiffness on the motile properties of myosin VI. Our results reveal that a stiff cargo–motor linkage leads to shorter step sizes and load-induced anchoring of myosin VI, while a flexible linkage results in longer steps with frequent detachments from the actin filament under load. Our findings suggest a novel regulatory mechanism for tuning the dual cellular roles of the anchor and transporter ascribed to myosin VI.

DOI

Mechanical Unfolding and Folding of a Complex Slipknot Protein Probed by Using Optical Tweezers

Han Wang, Xiaoqing Gao, Xiaodong Hu, Xiaotang Hu, Chunguang Hu, Hongbin Li

Knotted and slipknotted proteins are topologically complex. Understanding their folding and unfolding mechanism has attracted considerable interest. Here we combined protein engineering, single-molecule optical tweezers, and steered molecular dynamics (SMD) simulations to investigate the mechanical unfolding and folding of a slipknotted protein pyruvoyl-dependent arginine decarboxylase (PADC). In its slipknotted structure, PADC contains a long threaded loop (85 residues), which is almost twice the size of the knotting loop. When stretched from its N- and C-termini, the majority of PADC can be readily unfolded in a two-state manner, and the slipknotted structure was untied. A small percentage of PADC unfolded following a three-state pathway involving the formation of an unfolding intermediate state. These unfolding intermediate states showed a broad distribution of contour length increments, suggesting that they did not have a well-defined specific structure. SMD simulations revealed the main free energy barrier to the unfolding of PADC and suggested that the unfolding intermediate states may originate from the frication of polypeptide chain sliding during the process of pulling the threaded loop out of the knotting loop. Upon relaxation, a small percentage of the unfolded and untied PADC polypeptide chain can refold back to its native slipknotted conformation, but a large fraction can only reach a misfolded state. Our results revealed the complexity of the mechanical unfolding and refolding of a slipknotted protein with a long threaded loop.

DOI

A Disease-Causing Intronic Point Mutation C19G Alters Tau Exon 10 Splicing via RNA Secondary Structure Rearrangement

Jiazi Tan, Lixia Yang, Alan Ann Lerk Ong, Jiahao Shi, Zhensheng Zhong, Mun Leng Lye, Shiyi Liu, Jolanta Lisowiec-Wachnicka, Ryszard Kierzek, Xavier Roca, Gang Chen

Alternative splicing of MAPT cassette exon 10 produces tau isoforms with four microtubule-binding repeat domains (4R) upon exon inclusion or three repeats (3R) upon exon skipping. In human neurons, deviations from the ∼1:1 physiological 4R:3R ratio lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Certain FTDP-17-associated mutations affect a regulatory hairpin that sequesters the exon 10 5′ splice site (5′ss, located at the exon 10–intron 10 junction). These mutations tend to increase the 4R:3R ratio by destabilizing the hairpin, thereby improving 5′ss recognition by U1 snRNP. Interestingly, a single C-to-G mutation at the 19th nucleotide in intron 10 (C19G or +19G) decreases the level of exon 10 inclusion significantly from 56% to 1%, despite the disruption of a G-C base pair in the bottom stem of the hairpin. Here, we show by biophysical characterization, including thermal melting, fluorescence, and single-molecule mechanical unfolding using optical tweezers, that the +19G mutation alters the structure of the bottom stem, resulting in the formation of a new bottom stem with enhanced stability. The cell culture alternative splicing patterns of a series of minigenes reveal that the splicing activities of the mutants with destabilizing mutations on the top stem can be compensated in a position-dependent manner by the +19G mutation in the bottom stem. We observed an excellent correlation between the level of exon 10 inclusion and the rate of mechanical unfolding at 10 pN, indicating that the unfolding of the splice site hairpins (to facilitate subsequent binding of U1 snRNA) may be aided by helicases or other proteins.

DOI

Random Formation of G-Quadruplexes in the Full-Length Human Telomere Overhangs Leads to a Kinetic Folding Pattern with Targetable Vacant G-Tracts

Jibin Abraham Punnoose, Yue Ma, Mohammed Enamul Hoque, Yunxi Cui, Shogo Sasaki, Athena Huixin Guo, Kazuo Nagasawa, and Hanbin Mao

G-Quadruplexes formed in the 3′ telomere overhang (∼200 nucleotides) have been shown to regulate biological functions of human telomeres. The mechanism governing the population pattern of multiple telomeric G-quadruplexes is yet to be elucidated inside the telomeric overhang in a time window shorter than thermodynamic equilibrium. Using a single-molecule force ramping assay, we quantified G-quadruplex populations in telomere overhangs over a full physiological range of 99–291 nucleotides. We found that G-quadruplexes randomly form in these overhangs within seconds, which leads to a population governed by a kinetic, rather than a thermodynamic, folding pattern. The kinetic folding gives rise to vacant G-tracts between G-quadruplexes. By targeting these vacant G-tracts using complementary DNA fragments, we demonstrated that binding to the telomeric G-quadruplexes becomes more efficient and specific for telomestatin derivatives.

DOI

Quantification of Chemical and Mechanical Effects on the Formation of the G-Quadruplex and i-Motif in Duplex DNA

Sangeetha Selvam, Shankar Mandal, and Hanbin Mao

The formation of biologically significant tetraplex DNA species, such as G-quadruplexes and i-motifs, is affected by chemical (ions and pH) and mechanical [superhelicity (σ) and molecular crowding] factors. Because of the extremely challenging experimental conditions, the relative importance of these factors on tetraplex folding is unknown. In this work, we quantitatively evaluated the chemical and mechanical effects on the population dynamics of DNA tetraplexes in the insulin-linked polymorphic region using magneto-optical tweezers. By mechanically unfolding individual tetraplexes, we found that ions and pH have the largest effects on the formation of the G-quadruplex and i-motif, respectively. Interestingly, superhelicity has the second largest effect followed by molecular crowding conditions. While chemical effects are specific to tetraplex species, mechanical factors have generic influences. The predominant effect of chemical factors can be attributed to the fact that they directly change the stability of a specific tetraplex, whereas the mechanical factors, superhelicity in particular, reduce the stability of the competing species by changing the kinetics of the melting and annealing of the duplex DNA template in a nonspecific manner. The substantial dependence of tetraplexes on superhelicity provides strong support that DNA tetraplexes can serve as topological sensors to modulate fundamental cellular processes such as transcription.

DOI

Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin αIIbβ3

Olga Kononova, Rustem I. Litvinov, Dmitry S. Blokhin, Vladimir V. Klochkov, John W. Weisel, Joel S. Bennett, and Valeri Barsegov

Binding of soluble fibrinogen to the activated conformation of the integrin αIIbβ3 is required for platelet aggregation and is mediated exclusively by the C-terminal AGDV-containing dodecapeptide (γC-12) sequence of the fibrinogen γ chain. However, peptides containing the Arg-Gly-Asp (RGD) sequences located in two places in the fibrinogen Aα chain inhibit soluble fibrinogen binding to αIIbβ3 and make substantial contributions to αIIbβ3 binding when fibrinogen is immobilized and when it is converted to fibrin. Here, we employed optical trap-based nanomechanical measurements and computational molecular modeling to determine the kinetics, energetics, and structural details of cyclic RGDFK (cRGDFK) and γC-12 binding to αIIbβ3. Docking analysis revealed that NMR-determined solution structures of cRGDFK and γC-12 bind to both the open and closed αIIbβ3 conformers at the interface between the αIIb β-propeller domain and the β3 βI domain. The nanomechanical measurements revealed that cRGDFK binds to αIIbβ3 at least as tightly as γC-12. A subsequent analysis of molecular force profiles and the number of peptide−αIIbβ3 binding contacts revealed that both peptides form stable bimolecular complexes with αIIbβ3 that dissociate in the 60–120 pN range. The Gibbs free energy profiles of the αIIbβ3–peptide complexes revealed that the overall stability of the αIIbβ3-cRGDFK complex was comparable with that of the αIIbβ3−γC-12 complex. Thus, these results provide a mechanistic explanation for previous observations that RGD- and AGDV-containing peptides are both potent inhibitors of the αIIbβ3–fibrinogen interactions and are consistent with the observation that RGD motifs, in addition to AGDV, support interaction of αIIbβ3 with immobilized fibrinogen and fibrin.

DOI

Mutually Exclusive Formation of G-Quadruplex and i-Motif Is a General Phenomenon Governed by Steric Hindrance in Duplex DNA

Yunxi Cui, Deming Kong, Chiran Ghimire, Cuixia Xu, and Hanbin Mao

G-Quadruplex and i-motif are tetraplex structures that may form in opposite strands at the same location of a duplex DNA. Recent discoveries have indicated that the two tetraplex structures can have conflicting biological activities, which poses a challenge for cells to coordinate. Here, by performing innovative population analysis on mechanical unfolding profiles of tetraplex structures in double-stranded DNA, we found that formations of G-quadruplex and i-motif in the two complementary strands are mutually exclusive in a variety of DNA templates, which include human telomere and promoter fragments of hINS and hTERT genes. To explain this behavior, we placed G-quadruplex- and i-motif-hosting sequences in an offset fashion in the two complementary telomeric DNA strands. We found simultaneous formation of the G-quadruplex and i-motif in opposite strands, suggesting that mutual exclusivity between the two tetraplexes is controlled by steric hindrance. This conclusion was corroborated in the BCL-2 promoter sequence, in which simultaneous formation of two tetraplexes was observed due to possible offset arrangements between G-quadruplex and i-motif in opposite strands. The mutual exclusivity revealed here sets a molecular basis for cells to efficiently coordinate opposite biological activities of G-quadruplex and i-motif at the same dsDNA location.

DOI

Intermediates Stabilized by Tryptophan Pairs Exist in Trpzip Beta-Hairpins

Zhongbo Yu, Sangeetha Selvam, and Hanbin Mao
Transitions of protein secondary structures, such as alpha-helices and beta-hairpins, are often too small and too fast to follow by many single-molecular approaches. Here we describe new population deconvolution methods to investigate the mechanical unfolding/refolding events in Trpzip β-hairpins that are tethered between two optically trapped polystyrene particles through click chemistry. The application of force to the Trpzip peptides shifted population distribution, which allowed us to identify intermediates from regular force–extension curves of the peptides after population deconvolution analysis. Comparison of the intermediates between the Trpzip2 and Trpzip4 peptides suggests the intermediates are likely stabilized by the tryptophan pair stacking. We anticipate the method of population deconvolution described here can offer a unique capability to investigate fast transitions in small biological structures.

DOI

Analysis of diffuse K+ and Mg2+ ion binding to a two-base-pair kissing complex by single-molecule mechanical unfolding

Pan TX Li

The folding and stability of RNA tertiary interactions depends critically on cationic conditions. It is usually difficult, however, to isolate such effects on tertiary interactions from those on the entire RNA. By manipulating conformations of single RNA molecules using optical tweezers, we distinguished individual steps of breaking and forming of a two-base-pair kissing interaction from those of secondary folding. The binding of metal ions to the small tertiary structure appeared to be saturable with an apparent Kd of 160 mM for K+ and 1.5 mM for Mg2+. The kissing formation was estimated to be associated with binding of ~2-3 diffuse K+ or Mg2+ ions. At their saturated binding, Mg2+ provided ~3 kcal/mol more stabilizing energy to the structure than K+. Furthermore, the cations change the unkissing forces significantly more than the kissing ones. For example, the presence of Mg2+ ions increased the average unkissing force from 21 pN to 44 pN, surprisingly high for breaking merely two base pairs; in contrast, the mean kissing force was changed by only 4.5 pN. Interestingly, the differential salt effects on the transition forces were not caused by different changes in the height of the kinetic barriers, but were instead attributed to how different molecular structures respond to the applied force. Our results showed the importance of diffuse cation binding to the stability of tertiary interaction and demonstrated the utility of mechanical unfolding in studying tertiary interactions.
DOI

Tight Hydrophobic Contacts with the SecB Chaperone Prevent Folding of Substrate Proteins

Philipp Bechtluft, Alexej Kedrov, Dirk-Jan Slotboom, Nico Nouwen, Sander J. Tans and Arnold J. M. Driessen

The molecular chaperone SecB binds to hydrophobic sections of unfolded secretory proteins and thereby prevents their premature folding prior to secretion by the translocase ofEscherichia coli. Here, we have investigated the effect of the single-residue mutation of leucine 42 to arginine (L42R) centrally positioned in the polypeptide binding pocket of SecB on its chaperonin function. The mutant retains its tetrameric structure and SecA targeting function but is defective in its holdase activity. Isothermal titration calorimetry and single-molecule optical tweezer studies suggest that the SecB(L42R) mutant exhibits a reduced polypeptide binding affinity allowing for partial folding of the bound polypeptide chain rendering it translocation-incompetent.

DOI