Paul J. Bujalowski

Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45-motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail.

Tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods

During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp and discuss prominent applications. We provide protocols for the construction of polyproteins which are amenable for AFM experiments, the preparation of different coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria for AFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of the obtained data.

UNC-45B Chaperone: The Role of its Domains in the Interaction with the Myosin Motor Domain

The proper folding of many proteins can only be achieved by interaction with molecular chaperones. The molecular chaperone UNC-45B is required for the folding of striated muscle myosin II. However, the precise mechanism by which it contributes to proper folding of the myosin head remains unclear. UNC-45B contains three domains: an N-terminal TPR domain known to bind Hsp90, a Central domain of unknown function, and a C-terminal UCS domain known to interact with the myosin head. Here we used fluorescence titrations methods, dynamic light scattering, and single-molecule atomic force microscopy (AFM) unfolding/refolding techniques to study the interactions of the UCS and Central domains with the myosin motor domain. We found that both the UCS and the Central domains bind to the myosin motor domain. Our data show that the domains bind to distinct subsites on the myosin head, suggesting distinct roles in forming the myosin-UNC-45B complex. To determine the chaperone activity of the UCS and Central domains, we used two different methods: 1), prevention of misfolding using single-molecule AFM, and 2), prevention of aggregation using dynamic light scattering. Using the first method, we found that the UCS domain is sufficient to prevent misfolding of a titin mechanical reporter. Application of the second method showed that the UCS domain but not the Central domain prevents the thermal aggregation of the myosin motor domain. We conclude that while both the UCS and the Central domains bind the myosin head with high affinity, only the UCS domain displays chaperone activity.

Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes STOICHIOMETRIES, INTRINSIC AFFINITIES, COOPERATIVITIES, BASE, AND CONFORMATIONAL SPECIFICITIES

Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and double-stranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ± 2 nucleotides. In the presence of Mg2+, the site size increases to 29 ± 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex.

Chaperone‐mediated reversible inhibition of the sarcomeric myosin power stroke

Molecular chaperones are required for successful folding and assembly of sarcomeric myosin in skeletal and cardiac muscle. Here, we show that the chaperone UNC‐45B inhibits the actin translocation function of myosin. Further, we show that Hsp90, another chaperone involved in sarcomere development, allows the myosin to resume actin translocation. These previously unknown activities may play a key role in sarcomere development, preventing untimely myosin powerstrokes from disrupting the precise alignment of the sarcomere until it has formed completely.

Thermally‐induced structural changes in an armadillo repeat protein suggest a novel thermosensor mechanism in a molecular chaperone

Molecular chaperones are commonly identified by their ability to suppress heat‐induced protein aggregation. The muscle‐specific molecular chaperone UNC‐45B is known to be involved in myosin folding and is trafficked to the sarcomeres A‐band during thermal stress. Here, we identify temperature‐dependent structural changes in the UCS chaperone domain of UNC‐45B that occur within a physiologically relevant heat‐shock range. We show that distinct changes to the armadillo repeat protein topology result in exposure of hydrophobic patches, and increased flexibility of the molecule. These rearrangements suggest the existence of a novel thermosensor within the chaperone domain of UNC‐45B. We propose that these changes may function to suppress aggregation under stress by allowing binding to a wide variety of aggregation prone loops on its client.

Single-Molecule Methods

Single-molecule methods have emerged as powerful tools in life science research. These techniques allow the detection and manipulation of individual biological molecules and investigate, with unprecedented resolution, their conformations and dynamics at the nanoscale level. These techniques overcome the restrictions of traditional bulk biochemical studies by focusing on individuals of molecules. Here we describe some of the most common single-molecule methods including atomic force microscopy, optical tweezers, and fluorescence microscopy. We also describe the use of cryo-electron microscopy methods to study large molecules and macromolecular assemblies. We outline the principles of operation for each technique and discuss prominent applications.

Analysis of the REJ Module of Polycystin-1 Using Molecular Modeling and Force-Spectroscopy Techniques

Polycystin-1 is a large transmembrane protein, which, when mutated, causes autosomal dominant polycystic kidney disease, one of the most common life-threatening genetic diseases that is a leading cause of kidney failure. The REJ (receptor for egg lelly) module is a major component of PC1 ectodomain that extends to about 1000 amino acids. Many missense disease-causing mutations map to this module; however, very little is known about the structure or function of this region. We used a combination of homology molecular modeling, protein engineering, steered molecular dynamics (SMD) simulations, and single-molecule force spectroscopy (SMFS) to analyze the conformation and mechanical stability of the first ~420 amino acids of REJ. Homology molecular modeling analysis revealed that this region may contain structural elements that have an FNIII-like structure, which we named REJd1, REJd2, REJd3, and REJd4. We found that REJd1 has a higher mechanical stability than REJd2 (~190 pN and 60 pN, resp.). Our data suggest that the putative domains REJd3 and REJd4 likely do not form mechanically stable folds. Our experimental approach opens a new way to systematically study the effects of disease-causing mutations on the structure and mechanical properties of the REJ module of PC1.

The central domain of UNC‐45 chaperone inhibits the myosin power stroke

The multidomain UNC‐45B chaperone is crucial for the proper folding and function of sarcomeric myosin. We recently found that UNC‐45B inhibits the translocation of actin by myosin. The main functions of the UCS and TPR domains are known but the role of the central domain remains obscure. Here, we show—using in vitro myosin motility and ATPase assays—that the central domain alone acts as an inhibitor of the myosin power stroke through a mechanism that allows ATP turnover. Hence, UNC‐45B is a unique chaperone in which the TPR domain recruits Hsp90; the UCS domain possesses chaperone‐like activities; and the central domain interacts with myosin and inhibits the actin translocation function of myosin. We hypothesize that the inhibitory function plays a critical role during the assembly of myofibrils under stress and during the sarcomere development process.

Interactions between the dengue virus polymerase NS5 and stem-loop A

ABSTRACT The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5′ untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl2 but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA70 and SLA80, which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides. IMPORTANCE Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5′ untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the energetics of intrinsic affinities, characterize the stoichiometry of the complex of NS5 and SLA, and determine how solution conditions such as magnesium and sodium concentrations and temperature influence NS5-SLA interactions in solution. Quantitatively characterizing dengue virus NS5-SLA interactions will facilitate the design and assessment of antiviral therapeutics that target this essential step of the dengue virus life cycle.