Allosteric opening of the polypeptide-binding site when an Hsp70 binds ATP

Ruifeng Qi, Evans Boateng Sarbeng, Qun Liu, Katherine Quynh Le,  Xinping Xu, Hongya Xu, Jiao Yang, Jennifer Li Wong, Christina Vorvis, Wayne A Hendrickson, Lei Zhou, and Qinglian Liu

The 70-kilodalton (kDa) heat-shock proteins (Hsp70s) are ubiquitous molecular chaperones essential for cellular protein folding and proteostasis. Each Hsp70 has two functional domains: a nucleotide-binding domain (NBD), which binds and hydrolyzes ATP, and a substrate-binding domain (SBD), which binds extended polypeptides. NBD and SBD interact little when in the presence of ADP; however, ATP binding allosterically couples the polypeptide- and ATP-binding sites. ATP binding promotes polypeptide release; polypeptide rebinding stimulates ATP hydrolysis. This allosteric coupling is poorly understood. Here we present the crystal structure of an intact ATP-bound Hsp70 from Escherichia coli at 1.96-Å resolution. The ATP-bound NBD adopts a unique conformation, forming extensive interfaces with an SBD that has changed radically, having its α-helical lid displaced and the polypeptide-binding channel of its β-subdomain restructured. These conformational changes, together with our biochemical assays, provide a structural explanation for allosteric coupling in Hsp70 activity.

DOI

Journal:Nature Structural & Molecular Biology

Defining Single Molecular Forces Required to Activate Integrin and Notch Signaling

Xuefeng Wang, Taekjip Ha

Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of about 40 piconewtons (pN) to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions.

DOI

Journal: Science

A Tissue-Like Printed Material

Gabriel Villar, Alexander D. Graham, and Hagan Bayley

Living cells communicate and cooperate to produce the emergent properties of tissues. Synthetic mimics of cells, such as liposomes, are typically incapable of cooperation and therefore cannot readily display sophisticated collective behavior. We printed tens of thousands of picoliter aqueous droplets that become joined by single lipid bilayers to form a cohesive material with cooperating compartments. Three-dimensional structures can be built with heterologous droplets in software-defined arrangements. The droplet networks can be functionalized with membrane proteins; for example, to allow rapid electrical communication along a specific path. The networks can also be programmed by osmolarity gradients to fold into otherwise unattainable designed structures. Printed droplet networks might be interfaced with tissues, used as tissue engineering substrates, or developed as mimics of living tissue.

DOI

Journal: Science

Non-optimal codon usage affects expression, structure and function of clock protein FRQ

Mian Zhou, Jinhu Guo, Joonseok Cha, Michael Chae, She Chen, Jose M. Barral, Matthew S. Sachs, and Yi Liu

Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes1, 2, 3. Codon usage is also implicated in the control of transcription, splicing and RNA structure4, 5, 6. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

DOI

Journal: Nature

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments

Helena Gradišar, Sabina Božič, Tibor Doles, Damjan Vengust, Iva Hafner-Bratkovič, Alenka Mertelj, Ben Webb, Andrej Šali, Sandi Klavžar, and Roman Jerala

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil–forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

DOI

Journal: Nature Chemical Biology

Torque Spectroscopy of DNA: Base-Pair Stability, Boundary Effects, Backbending, and Breathing Dynamics

Florian C. Oberstrass, Louis E. Fernandes, Paul Lebel, and Zev Bryant

Changes in global DNA linking number can be accommodated by localized changes in helical structure. We have used single-molecule torque measurements to investigate sequence-specific strand separation and Z-DNA formation. By controlling the boundary conditions at the edges of sequences of interest, we have confirmed theoretical predictions of distinctive boundary-dependent backbending patterns in torque-twist relationships. Abrupt torque jumps are associated with the formation and collapse of DNA bubbles, permitting direct observations of DNA breathing dynamics.

DOI

Journal: Physical Review Letters

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field

Kevin J. Freedman, S. Raza Haq, Joshua B. Edel, Per Jemth, and Min Jun Kim

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106 V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein..

DOI

Journal: Scientific Reports