Hydrodynamic and thermodynamic analysis of PEGylated human serum albumin.

Covalent labeling of therapeutic drugs and proteins with polyethylene glycol (PEGylation) is an important modification for improving stability, solubility, and half-life. PEGylation alters protein solution behavior through its impact on thermodynamic nonideality by increasing the excluded volume, and on hydrodynamic nonideality by increasing the frictional drag. To understand PEGylation's impact, we investigated the thermodynamic and hydrodynamic properties of a model system consisting of PEGylated human serum albumin derivatives using analytical ultracentrifugation (AUC) and dynamic light scattering (DLS). We constructed PEGylated human serum albumin derivatives of single, linear 5K, 10K, 20K, and 40K PEG chains and a single branched-chain PEG of 40K (2 × 20K). Sedimentation velocity (SV) experiments were analyzed using SEDANAL direct boundary fitting to extract ideal sedimentation coefficients so, hydrodynamic nonideality ks, and thermodynamic nonideality 2BM1SV terms. These quantities allow the determination of the Stokes radius Rs, the frictional ratio f/fo, and the swollen or entrained volume Vs/v, which measure size, shape, and solvent interaction. We performed sedimentation equilibrium experiments to obtain independent measurements of thermodynamic nonideality 2BM1SE. From DLS measurements, we determined the interaction parameter, kD, the concentration dependence of the apparent diffusion coefficient, D, and from extrapolation of D to c = 0 a second estimate of Rs. Rs values derived from SV and DLS measurements and ensemble model calculations (see complementary study) are then used to show that ks + kD = theoretical 2B22M1. In contrast, experimental BM1 values from SV and sedimentation equilibrium data collectively allow for similar analysis for protein-PEG conjugates and show that ks + kD = 1.02-1.07∗BM1, rather than the widely used ks + kD = 2BM1 developed for hard spheres. The random coil behavior of PEG dominates the colloidal properties of PEG-protein conjugates and exceeds the sum of a random coil and hard-sphere volume due to excess entrained water.

Regulation of Protein Kinase R by Epstein-Barr Virus EBER1 RNA.

Protein kinase R (PKR) is a key antiviral component of the innate immune pathway and is activated by viral double-stranded RNAs (dsRNAs). Adenovirus-associated RNA 1 (VAI) is an abundant, noncoding viral RNA that functions as a decoy by binding PKR but not inducing activation, thereby inhibiting the antiviral response. In VAI, coaxial stacking produces an extended helix that mediates high-affinity PKR binding but is too short to result in activation. Like adenovirus, Epstein-Barr virus produces high concentrations of a noncoding RNA, EBER1. Here, we compare interactions of PKR with VAI and EBER1 and present a structural model of EBER1. Both RNAs function as inhibitors of dsRNA-mediated PKR activation. However, EBER1 weakly activates PKR whereas VAI does not. PKR binds EBER1 more weakly than VAI. Assays at physiological ion concentrations indicate that both RNAs can accommodate two PKR monomers and induce PKR dimerization. A structural model of EBER1 was obtained using constraints derived from chemical structure probing and small-angle X-ray scattering experiments. The central stem of EBER1 coaxially stacks with stem loop 4 and stem loop 1 to form an extended RNA duplex of ∼32 bp that binds PKR and promotes activation. Our observations that EBER1 binds PKR much more weakly than VAI and exhibits weak PKR activation suggest that EBER1 is less well suited to function as an RNA decoy.

Structural Basis of Protein Kinase R Autophosphorylation.

The RNA-activated protein kinase, PKR, is a key mediator of the innate immunity response to viral infection. Viral double-stranded RNAs induce PKR dimerization and autophosphorylation. The PKR kinase domain forms a back-to-back dimer. However, intermolecular ( trans) autophosphorylation is not feasible in this arrangement. We have obtained PKR kinase structures that resolves this dilemma. The kinase protomers interact via the known back-to-back interface as well as a front-to-front interface that is formed by exchange of activation segments. Mutational analysis of the front-to-front interface support a functional role in PKR activation. Molecular dynamics simulations reveal that the activation segment is highly dynamic in the front-to-front dimer and can adopt conformations conducive to phosphoryl transfer. We propose a mechanism where back-to-back dimerization induces a conformational change that activates PKR to phosphorylate a "substrate" kinase docked in a front-to-front geometry. This mechanism may be relevant to related kinases that phosphorylate the eukaryotic initiation factor eIF2α.

Design of High-Affinity Metal-Controlled Protein Dimers.

The ability to precisely control protein complex formation has high utility in the expanding field of biomaterials. Driving protein-protein binding through metal-ligand bridging interactions is a promising method of achieving this goal. Furthermore, the capacity to precisely regulate both complex formation and dissociation enables additional control not available with constitutive protein complexes. Here we describe the design of three metal-controlled protein dimers that are completely monomeric in the absence of metal yet form high-affinity symmetric homodimers in the presence of zinc sulfate. The scaffold used for the designed dimers is the ß1 domain of streptococcal protein G. In addition to forming high-affinity dimers in the presence of metal, the complexes also dissociate upon addition of EDTA. Biophysical characterization revealed that the proteins maintain relatively high thermal stability, bind with high affinity, and are completely monodisperse in the monomeric and dimeric states. High-resolution crystal structures revealed that the dimers adopt the target structure and that the designed metal-binding histidine residues successfully bind zinc and function to drive dimer formation.

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.

Effect of Aggregation on the Hydrodynamic Properties of Bovine Serum Albumin.

PURPOSE: To systematically analyze shape and size of soluble irreversible aggregates and the effect of aggregate formation on viscosity. METHODS: Online light scattering, refractive index and viscosity detectors attached to HPLC (Viscotek®) were used to study aggregation, molecular weight and intrinsic viscosity of bovine serum albumin (BSA). Irreversible aggregates were generated by heat stress. Bulk viscosity was measured by an oscillating piston viscometer. RESULTS: As BSA was heated at a higher concentration or for a longer time, the relative contribution, molecular weight and intrinsic viscosity of aggregate species increased. Molecular shape was evaluated from intrinsic viscosity values, and aggregates were estimated to be more asymmetric than monomer species. The presence of aggregates resulted in an increase in bulk viscosity when relative contribution of very high molecular weight species exceeded 10%. CONCLUSIONS: For model system and conditions studied, generation of higher order aggregate species was concluded to be associated with an increase in molecular asymmetry. Elevated viscosity in the presence of aggregated species points to molecular asymmetry being a critical parameter affecting solution viscosity of BSA.

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.

Role of the Interdomain Linker in RNA-Activated Protein Kinase Activation.

RNA-activated protein kinase (PKR) is a key component of the interferon-induced antiviral pathway in higher eukaryotes. Upon recognition of viral dsRNA, PKR is activated via dimerization and autophosphorylation. PKR contains two N-terminal dsRNA binding domains (dsRBD) and a C-terminal kinase domain. The dsRBDs and the kinase are separated by a long, unstructured ∼80-amino acid linker in the human enzyme. The length of the N-terminal portion of the linker varies among PKR sequences, and it is completely absent in one ortholog. Here, we characterize the effects of deleting the variable region from the human enzyme to produce PKRΔV. The linker deletion results in quantitative but not qualitative changes in catalytic activity, RNA binding, and conformation. PKRΔV is somewhat more active and exhibits more cooperative RNA binding. As we previously observed for the full-length enzyme, PKRΔV is flexible in solution and adopts a range of compact and extended conformations. The conformational ensemble is biased toward compact states that might be related to weak interactions between the dsRBD and kinase domains. PKR retains RNA-induced autophosphorylation upon complete removal of the linker, indicating that the C-terminal, basic region is also not required for activity.

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.

Structural analysis of adenovirus VAI RNA defines the mechanism of inhibition of PKR.

Protein kinase R (PKR) is activated by dsRNA produced during virus replication and plays a major role in the innate immunity response to virus infection. In response, viruses have evolved multiple strategies to evade PKR. Adenovirus virus-associated RNA-I (VAI) is a short, noncoding transcript that functions as an RNA decoy to sequester PKR in an inactive state. VAI consists of an apical stem-loop, a highly structured central domain, and a terminal stem. Chemical probing and mutagenesis demonstrate that the central domain is stabilized by a pseudoknot. A structural model of VAI was obtained from constraints derived from chemical probing and small angle x-ray scattering (SAXS) measurements. VAI adopts a flat, extended conformation with the apical and terminal stems emanating from a protuberance in the center. This model reveals how the apical stem and central domain assemble to produce an extended duplex that is precisely tuned to bind a single PKR monomer with high affinity, thereby inhibiting activation of PKR by viral dsRNA.

Regulation of PKR by RNA: formation of active and inactive dimers.

PKR is a member of the eIF2α family of protein kinases that inhibit translational initiation in response to stress stimuli and functions as a key mediator of the interferon-induced antiviral response. PKR contains a dsRNA binding domain that binds to duplex regions present in viral RNAs, resulting in kinase activation and autophosphorylation. An emerging theme in the regulation of protein kinases is the allosteric linkage of dimerization and activation. The PKR kinase domain forms a back-to-back parallel dimer that is implicated in activation. We have developed a sensitive homo-Förster resonance energy transfer assay for kinase domain dimerization to directly probe the relationship among RNA binding, activation, and dimerization. In the case of perfect duplex RNAs, dimerization is correlated with activation and dsRNAs containing 30 bp or more efficiently induce kinase domain dimerization and activation. However, more complex duplex RNAs containing a 10-15 bp 2'-O-methyl RNA barrier produce kinase dimers but do not activate. Similarly, inactivating mutations within the PKR dimer interface that disrupt key electrostatic and hydrogen binding interactions fail to abolish dimerization. Our data support a model in which activating RNAs induce formation of a back-to-back parallel PKR kinase dimer whereas nonactivating RNAs either fail to induce dimerization or produce an alternative, inactive dimer configuration, providing an additional mechanism for distinguishing between host and pathogen RNA.

Analysis of SecA dimerization in solution.

The Sec pathway mediates translocation of protein across the inner membrane of bacteria. SecA is a motor protein that drives translocation of preprotein through the SecYEG channel. SecA reversibly dimerizes under physiological conditions, but different dimer interfaces have been observed in SecA crystal structures. Here, we have used biophysical approaches to address the nature of the SecA dimer that exists in solution. We have taken advantage of the extreme salt sensitivity of SecA dimerization to compare the rates of hydrogen-deuterium exchange of the monomer and dimer and have analyzed the effects of single-alanine substitutions on dimerization affinity. Our results support the antiparallel dimer arrangement observed in one of the crystal structures of Bacillus subtilis SecA. Additional residues lying within the preprotein binding domain and the C-terminus are also protected from exchange upon dimerization, indicating linkage to a conformational transition of the preprotein binding domain from an open to a closed state. In agreement with this interpretation, normal mode analysis demonstrates that the SecA dimer interface influences the global dynamics of SecA such that dimerization stabilizes the closed conformation.

The α C helix is a central regulator of PKR activation.

Protein kinase R (PKR) functions in the eukaryotic innate immune system as a first-line defense against viral infections. PKR binds viral dsRNA, leading to autophosphorylation and activation. In its active state, PKR can phosphorylate its primary substrate, eIF2 α , which blocks initiation of translation in the infected cell. It has been established that PKR activation occurs when the kinase domain dimerizes in a back-to-back configuration. However, the mechanism by which dimerization leads to enzymatic activation is not fully understood. Here, we investigate the structural mechanistic basis and energy landscape for PKR activation, with a focus on the α C helix - a kinase activation and signal integration hub - using all-atom equilibrium and enhanced sampling molecular dynamics simulations. By employing window-exchange umbrella sampling, we compute free energy profiles of activation which show that back-to-back dimerization stabilizes a catalytically competent conformation of PKR. Key hydrophobic residues in the homodimer interface contribute to stabilization of the α C helix in an active conformation and the position of its glutamate residue. Using linear mutual information analysis, we analyze allosteric communication connecting the protomers' N-lobes and the α C helix dimer interface with the α C helix.

Defining the Escherichia coli SecA dimer interface residues through in vivo site-specific photo-cross-linking.

The motor protein SecA is a core component of the bacterial general secretory (Sec) pathway and is essential for cell viability. Despite evidence showing that SecA exists in a dynamic monomer-dimer equilibrium favoring the dimeric form in solution and in the cytoplasm, there is considerable debate as to the quaternary structural organization of the SecA dimer. Here, a site-directed photo-cross-linking technique was utilized to identify residues on the Escherichia coli SecA (ecSecA) dimer interface in the cytosol of intact cells. The feasibility of this method was demonstrated with residue Leu6, which is essential for ecSecA dimerization based on our analytical ultracentrifugation studies of SecA L6A and shown to form the cross-linked SecA dimer in vivo with p-benzoyl-phenylalanine (pBpa) substituted at position 6. Subsequently, the amino terminus (residues 2 to 11) in the nucleotide binding domain (NBD), Phe263 in the preprotein binding domain (PBD), and Tyr794 and Arg805 in the intramolecular regulator of the ATPase 1 domain (IRA1) were identified to be involved in ecSecA dimerization. Furthermore, the incorporation of pBpa at position 805 did not form a cross-linked dimer in the SecA Δ2-11 context, indicating the possibility that the amino terminus may directly contact Arg805 or that the deletion of residues 2 to 11 alters the topology of the naturally occurring ecSecA dimer.

Role of the PAS sensor domains in the Bacillus subtilis sporulation kinase KinA.

Histidine kinases are sophisticated molecular sensors that are used by bacteria to detect and respond to a multitude of environmental signals. KinA is the major histidine kinase required for initiation of sporulation upon nutrient deprivation in Bacillus subtilis. KinA has a large N-terminal region (residues 1 to 382) that is uniquely composed of three tandem Per-ARNT-Sim (PAS) domains that have been proposed to constitute a sensor module. To further enhance our understanding of this "sensor" region, we defined the boundaries that give rise to the minimal autonomously folded PAS domains and analyzed their homo- and heteroassociation properties using analytical ultracentrifugation, nuclear magnetic resonance (NMR) spectroscopy, and multiangle laser light scattering. We show that PAS(A) self-associates very weakly, while PAS(C) is primarily a monomer. In contrast, PAS(B) forms a stable dimer (K(d) [dissociation constant] of <10 nM), and it appears to be the main N-terminal determinant of KinA dimerization. Analysis of KinA mutants deficient for one or more PAS domains revealed a critical role for PAS(B), but not PAS(A), in autophosphorylation of KinA. Our findings suggest that dimerization of PAS(B) is important for keeping the catalytic domain of KinA in a functional conformation. We use this information to propose a model for the structure of the N-terminal sensor module of KinA.

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.

Cytokinesis in Trypanosoma brucei relies on an orphan kinesin that dynamically crosslinks microtubules.

Many single-celled eukaryotes have complex cell morphologies defined by microtubules arranged into higher-order structures. The auger-like shape of the parasitic protist Trypanosoma brucei (T. brucei) is mediated by a parallel array of microtubules that underlies the plasma membrane. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the ingressing cleavage furrow and is essential for the completion of cytokinesis. We have characterized the biophysical properties of a truncated KLIF construct in vitro to gain mechanistic insight into the function of this novel kinesin. We find that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked by KLIF in an antiparallel orientation are translocated relative to one another, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we find that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new cell posterior, which is an essential requirement for the completion of cytokinesis in T. brucei.

Analysis of high-affinity binding of protein kinase R to double-stranded RNA.

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to double-stranded RNA (dsRNA). Our previous analysis of binding of PKR to dsRNAs ranging from 20 to 40 bp supports a dimerization model for activation in which 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high-affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt concentrations but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability of shorter dsRNAs to bind two PKR monomers is not sufficient to induce autophosphorylation. We propose that activation of PKR by longer RNAs is correlated with an alternative binding mode in which both of the dsRNA binding motifs contact the RNA, inducing PKR to dimerize via a direct interaction of the kinase domains.

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.

Activation of PKR: an open and shut case?

The double-stranded (ds) RNA-activated protein kinase, PKR, has a key role in the innate immunity response to viral infection in higher eukaryotes. PKR contains an N-terminal dsRNA-binding domain and a C-terminal kinase domain. In the prevalent autoinhibition model for PKR activation, dsRNA binding induces a conformational change that leads to the release of the dsRNA-binding domain from the kinase, thus relieving the inhibition of the latent enzyme. Structural and biophysical data now favor a model whereby dsRNA principally functions to induce dimerization of PKR via the kinase domain. This dimerization model has implications for other PKR regulatory mechanisms and represents a new structural paradigm for control of protein kinase activity.