Michael Hare

The Intermediate Chain of Cytoplasmic Dynein Is Partially Disordered and Gains Structure upon Binding to Light-Chain LC8 †

The N-terminal domain of dynein intermediate chain, IC(1-289), is highly disordered, but upon binding to dynein light-chain LC8, it undergoes a significant conformational change to a more ordered structure. Using circular dichroism and fluorescence spectroscopy, we demonstrate that the change in conformation is due to an increase in the helical structure and to enhanced compactness in the environment of tryptophan 161. An increase in helical structure and compactness is also observed with trimethylamine-N-oxide (TMAO), a naturally occurring osmolyte used here as a probe to identify regions with a propensity for induced folding. Global protection of IC(1-289) from protease digestion upon LC8 binding was localized to a segment that includes residues downstream of the LC8-binding site. Several smaller constructs of IC(1-289) containing the LC8-binding site and one of the predicted helix or coiled-coil segments were made. IC(1-143) shows no increase in helical structure upon binding, while IC(114-260) shows an increase in helical structure similar to what is observed with IC(1-289). Binding of IC(114-260) to LC8 was monitored by fluorescence and native gel electrophoresis and shows saturation of binding, a stoichiometry of 1:1, and moderate binding affinity. The induced folding of IC(1-289) upon LC8 binding suggests that LC8 could act through the intermediate chain to facilitate dynein assembly or regulate cargo-binding interactions.

Conformational dynamics promote binding diversity of dynein light chain LC8

A highly conserved and ubiquitous protein known as LC8 binds over twenty different partners, characteristic of a molecular hub (Barbar, 2008 Biochemistry, 47, 503-508). Structural studies of LC8 complexes with binding partners having diverse recognition sequences show that the same binding groove of LC8 accommodates the various binding motifs. Here we use thermodynamics and dynamics measurements of LC8 complexes to group LC8 binding partners in two categories: those whose binding is enthalpically driven and those that are entropically favored. Peptides that are enthalpically driven completely silence the millisecond-microsecond relaxation signal, suggesting a significant rigidifying of the binding groove, while peptides in the entropically favored group exhibit the same conformational dynamics as the free protein, suggesting that the peptide sits loosely in the binding groove and so retains flexibility of the groove, and presumably of the bound peptide. The inherent disorder in the LC8 binding groove and in LC8 binding partners allows both types of binding, accounts for the lack of a conserved recognition consensus motif and underlies the binding specificity and broad selectivity observed in LC8 binding.

Structural Features of LC8-Induced Self-Association of Swallow

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

Acetamide enolate: formation, reactivity, and proton affinity

Abstract Acetamide enolate ( 1 ) was selectively prepared in a Fourier transform mass spectrometer and a variable temperature flowing afterglow apparatus by the fluoride-induced desilylation of 2-(trimethylsilyl)acetamide. Its reactivity, proton affinity, and collision-induced dissociation spectra were explored and contrasted to its isomeric amidate anion ( 2 ). Since 1 and 2 are ambident nucleophiles, their reactivity with perfluoropropylene and perfluorobenzene was investigated. The unimolecular isomerization of 1 to 2 also was examined at temperatures up to 300 °C. No rearrangement was observed under these conditions indicating that the activation barrier is at least 32 kcal mol −1 . Structures and energies of acetamide, its conjugate bases, and the transition structure interconverting 1 and 2 were computed using a variety of ab initio and density functional theory approaches.

The gas-phase acidity of cyclopropene and simple alkyl derivatives: can they be measured?

Abstract The gas-phase acidity of 3-methylcyclopropene ( 5 ) at the allylic position was explored computationally and experimentally. G2+ calculations indicate that Δ H ° acid = 415.5 kcal/mol making 3-methyl-3-cyclopropenyl anion ( 6 ) an extremely strong base. This species is also predicted to be unstable with respect to electron loss (EA(3-methyl-3-cyclopropenyl radical) = −1.54 kcal/mol). A kinetic approach for determining the acidity of 5 using the hydroxide-induced desilylation of 3-methyl-3-trimethylsilylcyclopropene (the DePuy method) was employed but fails in this case because of an unanticipated rearrangement. This raises the question: Can the acidity of cyclopropene and its simple alkyl derivatives be measured? Positive and negative responses to this question are given and discussed.

Interactions of LC8 with N-Terminal Segments of the Intermediate Chain of Cytoplasmic Dynein

LC8, a highly conserved 10-kDa light chain, and IC74, a 74-kDa intermediate chain, are presumed to promote the assembly of the cytoplasmic dynein motor protein complex and to be engaged in the controlled binding and release of cargo. The interactions of LC8 from Drosophila melanogaster with constructs of IC74 were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Previously, we have performed limited proteolysis on the N-terminal domain of IC74, IC(1-289), when free and when bound to dynein light chains LC8 and Tctex-1[1]. We have also shown that upon addition of LC8, IC(1-289) undergoes a significant conformational change from a largely unfolded to a more ordered structure. The purpose of the work presented here is to determine whether residues 1-30 in IC74, predicted to be in a coiled coil, are involved in the stabilization of the protein upon binding to LC8. Constructs of IC74, IC(1-143), and IC(30-143) that include the LC8 binding site but with and without the first 30 residues were prepared, and their binding and protection patterns were compared to our previous results for IC(1-289). The results suggest that coiled coil residues 1-30 are not responsible for the increase in structure we observe when IC(1-289) binds to LC8.

Protein Interactions Within the Cytoplasmic Dynein Complex as Probed by Mass Spectrometry and Nmr

INTRODUCTION. Cytoplasmic dynein is a principle motor for minus-end directed transport along microtubules[1]. It functions to position organelles and other materials within the cell, and plays an essential role in the separation of chromosomes. Cytoplasmic dynein is a large (1.2 MDa) complex made up of two heavy chain subunits linked by stalk-like domains to a common base. The heavy chains contain the ATP hydrolysis and microtubule binding sites required for motility. Located at the base are a number of subunits thought to regulate the assembly of the complex and its attachment to appropriate cargo. The intermediate chain IC74 forms a key intermediary in the complex, as it is known to associate with the heavy chain and the essential accessory complex dynactin as well as other subunits in the base. We have found that two light chains, LC8 and Tctex-1, attach to the N-terminal domain of IC74 (N-IC74). Here, the interactions between IC74 and the light chains have been characterized by limited proteolysis and mass spectrometry and by NMR spectroscopy. METHODS. The interactions of LC8, Tctex-1 and the N-IC74 from Drosophila melanogaster were characterized in vitro by affinity methods, limited proteolysis, and circular dichroism spectroscopy. Limited digestion of N-IC74 in the presence and absence of LC8, Tctex-1 or both localized binding of the light and intermediate chains to specific segments in the protein. These interactions were further investigated by heteronuclear NMR. RESULTS. Limited tryptic digestion and mass spectrometry[2] localized binding of Tctex-1 to the vicinity of K104 and K105, and localized binding of LC8 to the region downstream of K130. Circular dichroism, fluorescence, sedimentation velocity and proteolysis studies indicate that NIC74 has limited secondary and tertiary structure at near physiological solution conditions. Upon addition of LC8, N-IC74 becomes more ordered. This conformational change is reflected in increased global protection of N-IC74 from proteolytic digestion following the interaction, and in a significant change in the CD signal consistent with a shift from random coil to a more helical conformation. The sequence of IC74 that is responsible for the increase in structure upon binding was identified by heteronuclear NMR using IC74 constructs that contain the binding site, and regions of the protein predicted to fold into a coiled coil structure.

Dynamics and stability of partially folded and unfolded BPTI analogs

We have used NMR for characterization of partially folded and unfolded ensembles of bovine pancreatic trypsin inhibitor (BPTI) that model the early stages of folding. A chemically synthesized analog of BPTI, [14-38]Abu, which has a single disulfide bond between loop residues 14 and 38 and the other four cysteines replaced by -amino-nbutyric acid (Abu), is an ensemble of multiple partially folded conformations in slow exchange at low temperature (1-10°C) and pH > 4.5 [1]. Native-like conformations are more favored in the core than in the rest of the molecule. At lower pH and elevated temperature, the structure in the core is lost to form an unfolded ensemble of conformations with no secondary structure detected by CD or NMR [2,3]. Selective labeling with allows for characterization of conformations in slow exchange. In the present work, we have determined the stability of individual segments of the partially folded [14-38]Abu using chemical denaturation and NMR. There is a significant difference in the urea unfolding profiles of residues in the core and those in the rest of the molecules. This shows that global unfolding is non-cooperative, with the core being the last to unfold, and that the most ordered structure is in the hydrophobic core of the native protein.