SlyD Accelerates trans-to-cis Prolyl Isomerization in a Mechanosignaling Protein under Load.

Prolyl isomerization is recognized as one of the key regulatory mechanisms, which plays a crucial role in cell signaling, ion channel gating, phage virus infection, and molecular timing. This isomerization is usually slow but often accelerated by an enzyme, called peptidyl-prolyl isomerase (PPIase). In the current project, we investigate using single-molecule force spectroscopy (SMFS) the impact of a bacterial PPIase, SlyD, on the cis-trans isomerization of the proline 2225 (P2225) in an isolated 20th domain of a cytoskeletal mechanosensing protein filamin-A (FlnA20). To explore the FlnA20-PPIase interaction, we have used multiple SMFS modes, like constant velocity, constant distance, and jumping trap experiments. In our previous study, we reported the unique nature of the P2225, which is conserved in all naturally occurring filamins and can slowly (minutes) interconvert between cis-trans isomers, in absence of any PPIase. Our current results show a staggering 25-fold acceleration of the trans-to-cis isomerization rate in the presence of saturating SlyD concentration (7.25 µM) compared to the unenzymatic condition. A SlyD concentration-dependent depletion of the trans isomeric lifetime was also observed. Additionally, we observed that SlyD stabilizes the cis-isomer in the native state of FlnA20 by ∼2 kBT. This is the first single-molecule observation of the cis-trans isomerization catalysis by a PPIase in a mechanosensing protein.

An Active, Ligand-Responsive Pulling Geometry Reports on Internal Signaling between Subdomains of the DnaK Nucleotide-Binding Domain in Single-Molecule Mechanical Experiments.

Single-molecule mechanical experiments have proven to be ideal tools for probing the energetics and mechanics of large proteins and domains. In this paper, we investigate the nucleotide-dependent unfolding mechanics of the nucleotide-binding domain (NBD) of the Hsp70 chaperone DnaK. The NBD binds ADP or ATP in the binding cleft formed by lobe I and lobe II, which consists of two subdomains each. When force is applied to the termini of the NBD, the observed unfolding forces are independent of the nucleotide state. In contrast, when force is applied across the nucleotide-binding pocket, the unfolding forces report specifically on the nucleotide-phosphate state. In this active, ligand-responsive pulling geometry, we observed a bifurcation of the unfolding pathway; the pathway proceeds either through a cooperative "coupled pathway" or through a noncooperative "uncoupled pathway". The partitioning between individual unfolding pathways can be effectively tuned by mutation or by the nucleotide exchange factor GrpE, i.e., by the factors affecting the strength of the lobe I-lobe II interactions within the native NBD. These experiments provide important insight into the molecular origin of the internal signaling between the subdomains of the nucleotide-binding domain of Hsp70 proteins and how signals are efficiently transferred inside the protein molecule.

Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor.

The glucocorticoid receptor (GR) is a prominent nuclear receptor linked to a variety of diseases and an important drug target. Binding of hormone to its ligand binding domain (GR-LBD) is the key activation step to induce signaling. This process is tightly regulated by the molecular chaperones Hsp70 and Hsp90 in vivo. Despite its importance, little is known about GR-LBD folding, the ligand binding pathway, or the requirement for chaperone regulation. In this study, we have used single-molecule force spectroscopy by optical tweezers to unravel the dynamics of the complete pathway of folding and hormone binding of GR-LBD. We identified a "lid" structure whose opening and closing is tightly coupled to hormone binding. This lid is located at the N terminus without direct contacts to the hormone. Under mechanical load, apo-GR-LBD folds stably and readily without the need of chaperones with a folding free energy of [Formula: see text] The folding pathway is largely independent of the presence of hormone. Hormone binds only in the last step and lid closure adds an additional [Formula: see text] of free energy, drastically increasing the affinity. However, mechanical double-jump experiments reveal that, at zero force, GR-LBD folding is severely hampered by misfolding, slowing it to less than 1·s-1 From the force dependence of the folding rates, we conclude that the misfolding occurs late in the folding pathway. These features are important cornerstones for understanding GR activation and its tight regulation by chaperones.

A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy.

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

α-Actinin/titin interaction: A dynamic and mechanically stable cluster of bonds in the muscle Z-disk.

Stable anchoring of titin within the muscle Z-disk is essential for preserving muscle integrity during passive stretching. One of the main candidates for anchoring titin in the Z-disk is the actin cross-linker α-actinin. The calmodulin-like domain of α-actinin binds to the Z-repeats of titin. However, the mechanical and kinetic properties of this important interaction are still unknown. Here, we use a dual-beam optical tweezers assay to study the mechanics of this interaction at the single-molecule level. A single interaction of α-actinin and titin turns out to be surprisingly weak if force is applied. Depending on the direction of force application, the unbinding forces can more than triple. Our results suggest a model where multiple α-actinin/Z-repeat interactions cooperate to ensure long-term stable titin anchoring while allowing the individual components to exchange dynamically.