Jeffrey W. Lary

Analytical Ultracentrifugation: Sedimentation Velocity and Sedimentation Equilibrium

Analytical ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution. AUC has broad applications for the study of biomacromolecules in a wide range of solvents and over a wide range of solute concentrations. Three optical systems are available for the analytical ultracentrifuge (absorbance, interference, and fluorescence) that permit precise and selective observation of sedimentation in real time. In particular, the fluorescence system provides a new way to extend the scope of AUC to probe the behavior of biological molecules in complex mixtures and at high solute concentrations. In sedimentation velocity (SV), the movement of solutes in high centrifugal fields is interpreted using hydrodynamic theory to define the size, shape, and interactions of macromolecules. Sedimentation equilibrium (SE) is a thermodynamic method where equilibrium concentration gradients at lower centrifugal fields are analyzed to define molecule mass, assembly stoichiometry, association constants, and solution nonideality. Using specialized sample cells and modern analysis software, researchers can use SV to determine the homogeneity of a sample and define whether it undergoes concentration-dependent association reactions. Subsequently, more thorough model-dependent analysis of velocity and equilibrium experiments can provide a detailed picture of the nature of the species present in solution and their interactions.

Mechanism of PKR Activation by dsRNA

Protein kinase R (PKR) is a central component of the interferon antiviral defense pathway. Upon binding double-stranded RNA (dsRNA), PKR undergoes autophosphorylation reactions that activate the kinase. PKR then phosphorylates eukaryotic initiation factor 2alpha, thus inhibiting protein synthesis in virally infected cells. Using a series of dsRNAs of increasing length, we define the mechanism of PKR activation. A minimal dsRNA of 30 bp is required to bind two PKR monomers and 30 bp is the smallest dsRNA that elicits autophosphorylation activity. Thus, the ability of dsRNAs to function as PKR activators is correlated with binding of two or more PKR monomers. Sedimentation velocity data fit a model where PKR monomers sequentially attach to a single dsRNA. These results support an activation mechanism where the role of the dsRNA is to bring two or more PKR monomers in close proximity to enhance dimerization via the kinase domain. This model explains the inhibition observed at high dsRNA concentrations and the strong dependence of maximum activation on dsRNA binding affinity. Binding affinities increase dramatically upon reducing the salt concentration from 200 to 75 mM NaCl and we observe that a second PKR can bind to the 20-bp dsRNA. Nonspecific assembly of PKR on dsRNA occurs stochastically without apparent cooperativity.

RNA dimerization promotes PKR dimerization and activation.

The double-stranded RNA (dsRNA)-activated protein kinase [protein kinase R (PKR)] plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15-bp dsRNA for one protein to bind and 30-bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eukaryotic initiation factor 2alpha, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15- to 30-bp limits: human immunodeficiency virus type 1 transactivation-responsive region (TAR) RNA, a 23-bp hairpin with three bulges that is known to dimerize. TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization to test whether RNA dimerization affects PKR dimerization and activation. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary-structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary-structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15- to 30-bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease.

Structure of CD84 provides insight into SLAM family function

The signaling lymphocyte activation molecule (SLAM) family includes homophilic and heterophilic receptors that modulate both adaptive and innate immune responses. These receptors share a common ectodomain organization: a membrane-proximal immunoglobulin constant domain and a membrane-distal immunoglobulin variable domain that is responsible for ligand recognition. CD84 is a homophilic family member that enhances IFN-γ secretion in activated T cells. Our solution studies revealed that CD84 strongly self-associates with a Kd in the submicromolar range. These data, in combination with previous reports, demonstrate that the SLAM family homophilic affinities span at least three orders of magnitude and suggest that differences in the affinities may contribute to the distinct signaling behavior exhibited by the individual family members. The 2.0 Å crystal structure of the human CD84 immunoglobulin variable domain revealed an orthogonal homophilic dimer with high similarity to the recently reported homophilic dimer of the SLAM family member NTB-A. Structural and chemical differences in the homophilic interfaces provide a mechanism to prevent the formation of undesired heterodimers among the SLAM family homophilic receptors. These structural data also suggest that, like NTB-A, all SLAM family homophilic dimers adopt a highly kinked organization spanning an end-to-end distance of ≈140 Å. This common molecular dimension provides an opportunity for all two-domain SLAM family receptors to colocalize within the immunological synapse and bridge the T cell and antigen-presenting cell.

Oligomerization of a MutS Mismatch Repair Protein from Thermus aquaticus

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization ofTaq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 μm. The ΔG 0 2–4 for the dimer to tetramer transition is approximately −6.9 ± 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

DOMAIN ARCHITECTURE AND BIOCHEMICAL CHARACTERIZATION OF VERTEBRATE MCM10

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase α-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase α-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.

The role of human Dicer-dsRBD in processing small regulatory RNAs.

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.

Are fluorescence-detected sedimentation velocity data reliable?

Sedimentation velocity analytical ultracentrifugation is a classical biophysical technique that is commonly used to analyze the size, shape, and interactions of biological macromolecules in solution. Fluorescence detection provides enhanced sensitivity and selectivity relative to the standard absorption and refractrometric detectors, but data acquisition is more complex and can be subject to interference from several photophysical effects. Here, we describe methods to configure sedimentation velocity measurements using fluorescence detection and evaluate the performance of the fluorescence optical system. The fluorescence detector output is linear over a concentration range of at least 1 to 500nM fluorescein and Alexa Fluor 488. At high concentrations, deviations from linearity can be attributed to the inner filter effect. A duplex DNA labeled with Alexa Fluor 488 was used as a standard to compare sedimentation coefficients obtained using fluorescence and absorbance detectors. Within error, the sedimentation coefficients agree. Thus, the fluorescence detector is capable of providing precise and accurate sedimentation velocity results that are consistent with measurements performed using conventional absorption optics, provided the data are collected at appropriate sample concentrations and the optics are configured correctly.

Heparin activates PKR by inducing dimerization.

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway. PKR is activated to undergo autophosphorylation upon binding to double-stranded RNAs or RNAs that contain duplex regions. Activated PKR phosphorylates the α subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis. PKR is also activated by heparin, a highly sulfated glycosaminoglycan. We have used biophysical methods to define the mechanism of PKR activation by heparin. Heparins as short as hexasaccharide bind strongly to PKR and activate autophosphorylation. In contrast to double-stranded RNA, heparin activates PKR by binding to the kinase domain. Analytical ultracentrifugation measurements support a thermodynamic linkage model where heparin binding allosterically enhances PKR dimerization, thereby activating the kinase. These results indicate that PKR can be activated by small molecules and represents a viable target for the development of novel antiviral agents.

Conformational Dynamics of the Rpt6 ATPase in Proteasome Assembly and Rpn14 Binding

Juxtaposed to either or both ends of the proteasome core particle (CP) can exist a 19S regulatory particle (RP) that recognizes and prepares ubiquitinated proteins for proteolysis. RP triphosphatase proteins (Rpt1-Rpt6), which are critical for substrate translocation into the CP, bind chaperone-like proteins (Hsm3, Nas2, Nas6, and Rpn14) implicated in RP assembly. We used NMR and other biophysical methods to reveal that S. cerevisiae Rpt6s C-terminal domain undergoes dynamic helix-coil transitions enabled by helix-destabilizing glycines within its two most C-terminal α helices. Rpn14 binds selectively to Rpt6s four-helix bundle, with surprisingly high affinity. Loss of Rpt6s partially unfolded state by glycine substitution (Rpt6 G³⁶⁰,³⁸⁷A) disrupts holoenzyme formation in vitro, an effect enhanced by Rpn14. S. cerevisiae lacking Rpn14 and incorporating Rpt6 G³⁶⁰,³⁸⁷A demonstrate hallmarks of defective proteasome assembly and synthetic growth defects. Rpt4 and Rpt5 exhibit similar exchange, suggesting that conserved structural heterogeneity among Rpt proteins may facilitate RP-CP assembly.