Probing Interdomain Linkers and Protein Supertertiary Structure In Vitro and in Live Cells with Fluorescent Protein Resonance Energy Transfer.

Many proteins are composed of independently-folded domains connected by flexible linkers. The primary sequence and length of such linkers can set the effective concentration for the tethered domains, which impacts rates of association and enzyme activity. The length of such linkers can be sensitive to environmental conditions, which raises questions as to how studies in dilute buffer relate to the highly-crowded cellular environment. To examine the role of linkers in domain separation, we measured Fluorescent Protein-Fluorescence Resonance Energy Transfer (FP-FRET) for a series of tandem FPs that varied in the length of their interdomain linkers. We used discrete molecular dynamics to map the underlying conformational distribution, which revealed intramolecular contact states that we confirmed with single molecule FRET. Simulations found that attached FPs increased linker length and slowed conformational dynamics relative to the bare linkers. This makes the CLYs poor sensors of inherent linker properties. However, we also showed that FP-FRET in CLYs was sensitive to solvent quality and macromolecular crowding making them potent environmental sensors. Finally, we targeted the same proteins to the plasma membrane of living mammalian cells to measure FP-FRET in cellulo. The measured FP-FRET when tethered to the plasma membrane was the same as that in dilute buffer. While caveats remain regarding photophysics, this suggests that the supertertiary conformational ensemble of these CLY proteins may not be affected by this specific cellular environment.

Activation of PKR by short stem-loop RNAs containing single-stranded arms.

Protein kinase R (PKR) is a central component of the innate immunity antiviral pathway and is activated by dsRNA. PKR contains a C-terminal kinase domain and two tandem dsRNA binding domains. In the canonical activation model, binding of multiple PKR monomers to dsRNA enhances dimerization of the kinase domain, leading to enzymatic activation. A minimal dsRNA of 30 bp is required for activation. However, short (∼15 bp) stem-loop RNAs containing flanking single-stranded tails (ss-dsRNAs) are capable of activating PKR. Activation was reported to require a 5'-triphosphate. Here, we characterize the structural features of ss-dsRNAs that contribute to activation. We have designed a model ss-dsRNA containing 15-nt single-stranded tails and a 15-bp stem and made systematic truncations of the tail and stem regions. Autophosphorylation assays and analytical ultracentrifugation experiments were used to correlate activation and binding affinity. PKR activation requires both 5'- and 3'-single-stranded tails but the triphosphate is dispensable. Activation potency and binding affinity decrease as the ssRNA tails are truncated and activation is abolished in cases where the binding affinity is strongly reduced. These results indicate that the single-stranded regions bind to PKR and support a model where ss-dsRNA induced dimerization is required but not sufficient to activate the kinase. The length of the duplex regions in several natural RNA activators of PKR is below the minimum of 30 bp required for activation and similar interactions with single-stranded regions may contribute to PKR activation in these cases.

The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin.

Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell surface receptors that increase the local toxin concentration on cell membranes, thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. Although it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell lines, we show that VCC is unlikely to utilize high affinity protein receptors as do structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding interactions.

Glycan specificity of the Vibrio vulnificus hemolysin lectin outlines evolutionary history of membrane targeting by a toxin family.

Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a ß-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH ß-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs.

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 Rpt6's 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 Rpt6's four-helix bundle, with surprisingly high affinity. Loss of Rpt6's partially unfolded state by glycine substitution (Rpt6 G³6°,³87A) disrupts holoenzyme formation in vitro, an effect enhanced by Rpn14. S. cerevisiae lacking Rpn14 and incorporating Rpt6 G³6°,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.

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.

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.

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.

Structure of the s5a:k48-linked diubiquitin complex and its interactions with rpn13.

Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin-interacting motifs (UIMs). Here, we use nuclear magnetic resonance (NMR) and analytical ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in which K48 of one ubiquitin subunit (the "proximal" one) is covalently bonded to G76 of the other (the "distal" subunit). We demonstrate that S5a's UIMs bind the two subunits simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13 bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is highly adaptive and cooperative toward binding ubiquitin chains.

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.

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.

Characterization of the neuron-specific L1-CAM cytoplasmic tail: naturally disordered in solution it exercises different binding modes for different adaptor proteins.

L1, a highly conserved transmembrane glycoprotein member of the immunoglobulin superfamily of cell adhesion molecules, mediates many developmental processes in the nervous system. Here we present the biophysical characterization and the binding properties of the least structurally defined part of this receptor: its cytoplasmic tail (CT). We have shown by analytical ultracentrifugation and dynamic light scattering experiments that it is mostly monomeric and unstructured in aqueous solution. We have defined by nuclear magnetic resonance the molecular details of L1-CT binding to two major targets: a membrane-cytoskeletal linker (MCL), ezrin, and an endocytosis mediator, AP2. Surprisingly, in addition to the two previously identified ezrin binding motifs, the juxtamembrane and the (1176)YRSLE regions, we have discovered a third one, a part of which has been previously associated with binding to another MCL, ankyrin. For the L1 interaction with AP2 we have determined the precise interaction region surrounding the (1176)YRSLE binding site and that this overlaps with the second ezrin binding site. In addition, we have shown that the juxtamembrane region of L1-CT has some binding affinity to AP2-mu2, although the specificity of this interaction needs further investigation. These data indicate that L1-CT belongs to the class of intrinsically disordered proteins. Endogenous flexibility of L1-CT might play an important role in dynamic regulation of intracellular signaling: the ability of cytoplasmic tails to accommodate different targets has the potential to fine-tune signal transduction via cell surface receptors.

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 alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.

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.

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-gamma secretion in activated T cells. Our solution studies revealed that CD84 strongly self-associates with a K(d) 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 A 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 approximately 140 A. 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.

Defining how ubiquitin receptors hHR23a and S5a bind polyubiquitin.

Ubiquitin receptors connect substrate ubiquitylation to proteasomal degradation. HHR23a binds proteasome subunit 5a (S5a) through a surface that also binds ubiquitin. We report that UIM2 of S5a binds preferentially to hHR23a over polyubiquitin, and we provide a model for the ternary complex that we expect represents one of the mechanisms used by the proteasome to capture ubiquitylated substrates. Furthermore, we demonstrate that hHR23a is surprisingly adept at sequestering the ubiquitin moieties of a polyubiquitin chain, and provide evidence that it and the ubiquitylated substrate are committed to each other after binding.

T cell immunoglobulin mucin-3 crystal structure reveals a galectin-9-independent ligand-binding surface.

The T cell immunoglobulin mucin (Tim) family of receptors regulates effector CD4(+) T cell functions and is implicated in autoimmune and allergic diseases. Tim-3 induces immunological tolerance, and engagement of the Tim-3 immunoglobulin variable (IgV) domain by galectin-9 is important for appropriate termination of T helper 1-immune responses. The 2 A crystal structure of the Tim-3 IgV domain demonstrated that four cysteines, which are invariant within the Tim family, form two noncanonical disulfide bonds, resulting in a surface not present in other immunoglobulin superfamily members. Biochemical and biophysical studies demonstrated that this unique structural feature mediates a previously unidentified galectin-9-independent binding process and suggested that this structural feature is conserved within the entire Tim family. The current work provided a graphic example of the relationship between sequence, structure, and function and suggested that the interplay between multiple Tim-3-binding activities contributes to the regulated assembly of signaling complexes required for effective Th1-mediated immunity.

Recognition of C-terminal amino acids in tubulin by pore loops in Spastin is important for microtubule severing.

Spastin, an AAA ATPase mutated in the neurodegenerative disease hereditary spastic paraplegia, severs microtubules. Many other AAA proteins form ring-shaped hexamers and contain pore loops, which project into the ring's central cavity and act as ratchets that pull on target proteins, leading, in some cases, to conformational changes. We show that Spastin assembles into a hexamer and that loops within the central pore recognize C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. We also show that Spastin contains a second microtubule binding domain that makes a distinct interaction with microtubules and is required for severing. Given that Spastin engages the MT in two places and that both interactions are required for severing, we propose that severing occurs by forces exerted on the C-terminal tail of tubulin, which results in a conformational change in tubulin, which releases it from the polymer.

NTB-A receptor crystal structure: insights into homophilic interactions in the signaling lymphocytic activation molecule receptor family.

The signaling lymphocytic activation molecule (SLAM) family includes homophilic and heterophilic receptors that regulate both innate and adaptive immunity. The ectodomains of most SLAM family members are composed of an N-terminal IgV domain and a C-terminal IgC2 domain. NK-T-B-antigen (NTB-A) is a homophilic receptor that stimulates cytotoxicity in natural killer (NK) cells, regulates bactericidal activities in neutrophils, and potentiates T helper 2 (Th2) responses. The 3.0 A crystal structure of the complete NTB-A ectodomain revealed a rod-like monomer that self-associates to form a highly kinked dimer spanning an end-to-end distance of approximately 100 A. The NTB-A homophilic and CD2-CD58 heterophilic dimers show overall structural similarities but differ in detailed organization and physicochemical properties of their respective interfaces. The NTB-A structure suggests a mechanism responsible for binding specificity within the SLAM family and imposes physical constraints relevant to the colocalization of SLAM-family proteins with other signaling molecules in the immunological synapse.

Mechanism of PKR activation: dimerization and kinase activation in the absence of double-stranded RNA.

The kinase PKR is a central component of the interferon antiviral pathway. PKR is activated upon binding double-stranded (ds) RNA to undergo autophosphorylation. Although PKR is known to dimerize, the relationship between dimerization and activation remains unclear. Here, we directly characterize dimerization of PKR in free solution using analytical ultracentrifugation and correlate self-association with autophosphorylation activity. Latent, unphosphorylated PKR exists predominantly as a monomer at protein concentrations below 2 mg/ml. A monomer sedimentation coefficient of s(20,w)(0)=3.58 S and a frictional ratio of f/f(0)=1.62 indicate an asymmetric shape. Sedimentation equilibrium measurements indicate that PKR undergoes a weak, reversible monomer-dimer equilibrium with K(d)=450 microM. This dimerization reaction serves to initiate a previously unrecognized dsRNA-independent autophosphorylation reaction. The resulting activated enzyme is phosphorylated on the two critical threonine residues present in the activation loop and is competent to phosphorylate the physiological substrate, eIF2alpha. Dimer stability is enhanced by approximately 500-fold upon autophosphorylation. We propose a chain reaction model for PKR dsRNA-independent activation where dimerization of latent enzyme followed by intermolecular phosphorylation serves as the initiation step. Subsequent propagation steps likely involve phosphorylation of latent PKR monomers by activated enzyme within high-affinity heterodimers. Our results support a model whereby dsRNA functions by bringing PKR monomers into close proximity in a manner that is analogous to the dimerization of free PKR.