Size Exclusion Chromatography-Mass Photometry: A New Method for Adeno-Associated Virus Product Characterization.

Over the past decade, adeno-associated viruses (AAVs) have attained significant prominence in gene therapy and genome editing applications, necessitating the development of robust and precise methodologies to ensure the quality and purity of AAV products. Existing AAV characterization techniques have proven effective for the analysis of pure and homogeneous AAV samples. However, there is still a demand for a rapid and low-sample-consumption method suitable for the characterization of lower purity or heterogeneous AAV samples commonly encountered in AAV products. Addressing this challenge, we propose the SEC-MP method, which combines size exclusion chromatography (SEC) with mass photometry (MP). In this novel approach, SEC effectively separates monomeric AAV particles from impurities, while the UV detector determines the virus particle concentration. MP complements this process by estimating the fraction of fully packaged AAVs in the total population of AAV particles. This combined methodology enables accurate determination of the titer of effective, fully packaged AAVs in samples containing aggregates, incorrectly packaged AAVs with incomplete genomes, protein or DNA fragments, and other impurities. Our experimental results demonstrate that SEC-MP provides valuable guidance for sample quality control and subsequent applications in the field of AAV research.

The dimeric form of bacterial l-asparaginase YpAI is fully active.

l-asparaginases from mesophilic bacteria (ASNases), including two enzymes very successfully used in the treatment of leukaemia, have been consistently described as homotetramers. On the contrary, structural studies show that homodimers of these enzymes should be sufficient to carry out the catalytic reaction. In this report, we investigated whether the type I Yersinia pestis asparaginase (YpAI) is active in a dimeric form or whether the tetrameric quaternary structure is critical for its activity. Using multiple biophysical techniques that investigate enzymatic properties and quaternary structure at either high or low protein concentration, we found that dimeric YpAI is fully active, suggesting that the tetrameric form of this subfamily of enzymes does not bear significant enzymatic relevance. In this process, we extensively characterized YpAI, showing that it is a cooperative enzyme, although the mechanism of allostery is still not definitely established. We showed that, like most type I ASNases, the substrate affinity of YpAI is low and this enzyme is very similar in terms of both the structure and enzymatic properties to homologous type I ASNase from Escherichia coli (EcAI). We extended these studies to more medically relevant type II ASNases, used as anti-leukaemia drugs. We confirmed that type II ASNases are not allosteric, and that they might also be functional in a dimeric form. However, the determination of the accurate tetramer⇆dimer dissociation constants of these enzymes that most likely lie in the picomolar range is not possible with currently available biophysical techniques.

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry.

Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high-resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations but also identify partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Sub-picomole quantities of sample are sufficient for MP analysis, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor the physical attributes of rAAV vectors.

Reformulation of an extant ATPase active site to mimic ancestral GTPase activity reveals a nucleotide base requirement for function.

Hydrolysis of nucleoside triphosphates releases similar amounts of energy. However, ATP hydrolysis is typically used for energy-intensive reactions, whereas GTP hydrolysis typically functions as a switch. SpoIVA is a bacterial cytoskeletal protein that hydrolyzes ATP to polymerize irreversibly during Bacillus subtilis sporulation. SpoIVA evolved from a TRAFAC class of P-loop GTPases, but the evolutionary pressure that drove this change in nucleotide specificity is unclear. We therefore reengineered the nucleotide-binding pocket of SpoIVA to mimic its ancestral GTPase activity. SpoIVAGTPase functioned properly as a GTPase but failed to polymerize because it did not form an NDP-bound intermediate that we report is required for polymerization. Further, incubation of SpoIVAGTPase with limiting ATP did not promote efficient polymerization. This approach revealed that the nucleotide base, in addition to the energy released from hydrolysis, can be critical in specific biological functions. We also present data suggesting that increased levels of ATP relative to GTP at the end of sporulation was the evolutionary pressure that drove the change in nucleotide preference in SpoIVA.

Living organisms need energy to stay alive; in cells, this energy is supplied in the form of a small molecule called adenosine triphosphate, or ATP, a nucleotide that stores energy in the bonds between its three phosphate groups. ATP is present in all living cells and is often referred to as the energy currency of the cell, because it can be easily stored and transported to where it is needed. However, it is unknown why cells rely so heavily on ATP when a highly similar nucleotide called guanosine triphosphate, or GTP, could also act as an energy currency. There are several examples of proteins that originally used GTP and have since evolved to use ATP, but it is not clear why this switch occurred. One suggestion is that ATP is the more readily available nucleotide in the cell. To test this hypothesis, Updegrove, Harke et al. studied a protein that helps bacteria transition into spores, which are hardier and can survive in extreme environments until conditions become favorable for bacteria to grow again. In modern bacteria, this protein uses ATP to provide energy, but it evolved from an ancestral protein that used GTP instead. First, Updegrove, Harke et al. engineered the protein so that it became more similar to the ancestral protein and used GTP instead of ATP. When this was done, the protein gained the ability to break down GTP and release energy from it, but it no longer performed its enzymatic function. This suggests that both the energy released and the source of that energy are important for a protein's activity. Further analysis showed that the modern version of the protein has evolved to briefly hold on to ATP after releasing its energy, which did not happen with GTP in the modified protein. Updegrove, Harke et al. also discovered that the levels of GTP in a bacterial cell fall as it transforms into a spore, while ATP levels remain relatively high. This suggests that ATP may indeed have become the source of energy of choice because it was more available. These findings provide insights into how ATP became the energy currency in cells, and suggest that how ATP is bound by proteins can impact a protein's activity. Additionally, these experiments could help inform the development of drugs targeting proteins that bind nucleotides: it may be essential to consider the entirety of the binding event, and not just the release of energy.

Chlorpromazine binding to the PAS domains uncovers the effect of ligand modulation on EAG channel activity.

Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per-Arnt-Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.

Investigating cyclic nucleotide and cyclic dinucleotide binding to HCN channels by surface plasmon resonance.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding domain (CNBD) in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides to the HCN pore opening. Recently, cyclic dinucleotides were shown to antagonize the effect of cyclic nucleotides in HCN4 but not in HCN2 channels. Based on the structural analysis and mutational studies it has been proposed that cyclic dinucleotides affect HCN4 channels by binding to the C-linker pocket (CLP). Here, we first show that surface plasmon resonance (SPR) can be used to accurately measure cyclic nucleotide binding affinity to the C-linker/CNBD of HCN2 and HCN4 channels. We then used SPR to investigate cyclic dinucleotide binding in HCN channels. To our surprise, we detected no binding of cyclic dinucleotides to the isolated monomeric C-linker/CNBDs of HCN4 channels with SPR. The binding of cyclic dinucleotides was further examined with isothermal calorimetry (ITC), which indicated no binding of cyclic dinucleotides to both monomeric and tetrameric C-linker/CNBDs of HCN4 channels. Taken together, our results suggest that interaction of the C-linker/CNBD with other parts of the channel is necessary for cyclic-dinucleotide binding in HCN4 channels.

Katanin spiral and ring structures shed light on power stroke for microtubule severing.

Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II.

Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLC-phosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts.

Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a ß-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded ß-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana, revealing three tandem polypeptide transport-associated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperone-like activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space.

Nonenzymatic conversion of ADP-ribosylated arginines to ornithine alters the biological activities of human neutrophil peptide-1.

Activated neutrophils, recruited to the airway of diseased lung, release human neutrophil peptides (HNP1-4) that are cytotoxic to airway cells as well as microbes. Airway epithelial cells express arginine-specific ADP ribosyltransferase (ART)-1, a GPI-anchored ART that transfers ADP-ribose from NAD to arginines 14 and 24 of HNP-1. We previously reported that ADP-ribosyl-arginine is converted nonenzymatically to ornithine and that ADP-ribosylated HNP-1 and ADP-ribosyl-HNP-(ornithine) were isolated from bronchoalveolar lavage fluid of a patient with idiopathic pulmonary fibrosis, indicating that these reactions occur in vivo. To determine effects of HNP-ornithine on the airway, three analogs of HNP-1, HNP-(R14orn), HNP-(R24orn), and HNP-(R14,24orn), were tested for their activity against Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus; their cytotoxic effects on A549, NCI-H441, small airway epithelial-like cells, and normal human lung fibroblasts; and their ability to stimulate IL-8 and TGF-ß1 release from A549 cells, and to serve as ART1 substrates. HNP and the three analogs had similar effects on IL-8 and TGF-ß1 release from A549 cells and were all cytotoxic for small airway epithelial cells, NCI-H441, and normal human lung fibroblasts. HNP-(R14,24orn), when compared with HNP-1 and HNP-1 with a single ornithine substitution for arginine 14 or 24, exhibited reduced cytotoxicity, but it enhanced proliferation of A549 cells and had antibacterial activity. Thus, arginines 14 and 24, which can be ADP ribosylated by ART1, are critical to the regulation of the cytotoxic and antibacterial effects of HNP-1. The HNP analog, HNP-(R14,24orn), lacks the epithelial cell cytotoxicity of HNP-1, but partially retains its antibacterial activity and thus may have clinical applications in airway disease.

An engineered palette of metal ion quenchable fluorescent proteins.

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.

Tubulin tyrosine ligase and stathmin compete for tubulin binding in vitro.

Tubulin partition between soluble and polymeric forms is tightly regulated in cells. Stathmin and tubulin tyrosine ligase (TTL) each form stable complexes with tubulin and inhibit tubulin polymerization. Here we explore the mutual relationship between these proteins in vitro and demonstrate that full-length stathmin and TTL compete for binding to tubulin and fail to make a stable tubulin:stathmin:TTL triple complex in solution. Moreover, stathmin depresses TTL tubulin tyrosination activity in vitro. These results suggest either that TTL and stathmin have a partially overlapping footprint on the tubulin dimer or that stathmin induces a tubulin conformation incompatible with stable TTL binding.

The maturational refolding of the ß-hairpin motif of equine infectious anemia virus capsid protein extends its helix α1 at capsid assembly locus.

A retroviral capsid (CA) protein consists of two helical domains, CA(N) and CA(C), which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a ß-hairpin motif upon processing from its precursor polyprotein Gag. The ß-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the ß-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the ß-hairpin structure demonstrated that folding of the ß-hairpin specifically extended the N terminus of helix α1 from Tyr(20) to Pro(17). This coil to helix transition involves the conserved sequence of Thr(16)-Pro(17)-Arg(18) (Ser(16)-Pro(17)-Arg(18) in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CA(N) oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg(18), helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the ß-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CA(N) oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CA(N) domain oligomerization, independent to the faster EIAV-CA(C) domain dimerization.

Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin.

Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of α-tubulin. Tyrosination regulates recruitment of microtubule-interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP grasp-fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms.

The crystal structure and mechanism of an unusual oxidoreductase, GilR, involved in gilvocarcin V biosynthesis.

GilR is a recently identified oxidoreductase that catalyzes the terminal step of gilvocarcin V biosynthesis and is a unique enzyme that establishes the lactone core of the polyketide-derived gilvocarcin chromophore. Gilvocarcin-type compounds form a small distinct family of anticancer agents that are involved in both photo-activated DNA-alkylation and histone H3 cross-linking. High resolution crystal structures of apoGilR and GilR in complex with its substrate pregilvocarcin V reveals that GilR belongs to the small group of a relatively new type of the vanillyl-alcohol oxidase flavoprotein family characterized by bicovalently tethered cofactors. GilR was found as a dimer, with the bicovalently attached FAD cofactor mediated through His-65 and Cys-125. Subsequent mutagenesis and functional assays indicate that Tyr-445 may be involved in reaction catalysis and in mediating the covalent attachment of FAD, whereas Tyr-448 serves as an essential residue initiating the catalysis by swinging away from the active site to accommodate binding of the 6R-configured substrate and consequently abstracting the proton of the hydroxyl residue of the substrate hemiacetal 6-OH group. These studies lay the groundwork for future enzyme engineering to broaden the substrate specificity of this bottleneck enzyme of the gilvocarcin biosynthetic pathway for the development of novel anti-cancer therapeutics.

Glutathionylation of peroxiredoxin I induces decamer to dimers dissociation with concomitant loss of chaperone activity.

Reversible protein glutathionylation, a redox-sensitive regulatory mechanism, plays a key role in cellular regulation and cell signaling. Peroxiredoxins (Prxs), a family of peroxidases that is involved in removing H(2)O(2) and organic hydroperoxides, are known to undergo a functional change from peroxidase to molecular chaperone upon overoxidation of its catalytic cysteine. The functional change is caused by a structural change from low molecular weight oligomers to high molecular weight complexes that possess molecular chaperone activity. We reported earlier that Prx I can be glutathionylated at three of its cysteine residues, Cys52, -83, and -173 [Park et al. (2009) J. Biol. Chem., 284, 23364]. In this study, using analytical ultracentrifugation analysis, we reveal that glutathionylation of Prx I, WT, or its C52S/C173S double mutant shifted its oligomeric status from decamers to a population consisting mainly of dimers. Cys83 is localized at the putative dimer-dimer interface, implying that the redox status of Cys83 may play an important role in stabilizing the oligomeric state of Prx I. Studies with the Prx I (C83S) mutant show that while Cys83 is not essential for the formation of high molecular weight complexes, it affects the dimer-decamer equilibrium. Glutathionylation of the C83S mutant leads to accumulation of dimers and monomers. In addition, glutathionylation of Prx I, both the WT and C52S/C173S mutants, greatly reduces their molecular chaperone activity in protecting citrate synthase from thermally induced aggregation. Together, these results reveal that glutathionylation of Prx I promotes changes in its quaternary structure from decamers to smaller oligomers and concomitantly inactivates its molecular chaperone function.

Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain.

NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.

Novel protective mechanism against irreversible hyperoxidation of peroxiredoxin: Nalpha-terminal acetylation of human peroxiredoxin II.

Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), cycles between thiol (C(P)-SH) and disulfide (-S-S-) states via a sulfenic (C(P)-SOH) intermediate. Hyperoxidation of the C(P) thiol to its sulfinic (C(P)-SO(2)H) derivative has been shown to be reversible, but its sulfonic (C(P)-SO(3)H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C(P)-SO(3)H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N(alpha)-terminal acetylation (N(alpha)-Ac) occurred exclusively on Prx II after demethionylation. N(alpha)-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N(alpha)-acetylated and N(alpha)-terminal acetylated Prx II revealed that N(alpha)-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T(m) from 59.6 to 70.9 degrees C. These findings suggest that the structural maintenance of Prx II by N(alpha)-Ac may be responsible for preventing its hyperoxidation to form C(P)-SO(3)H.

Solution NMR characterizations of oligomerization and dynamics of equine infectious anemia virus matrix protein and its interaction with PIP2.

Budding of retroviruses requires the structural precursor polyprotein, Gag, to target the plasma membrane through its N-terminal matrix (MA) domain. For HIV-1, the interaction between membrane signaling molecule phosphatidylinositol 4,5-diphosphate (PIP2) and MA induces the exposure of myristate and promotes membrane binding. Here we studied oligomerization of the naturally unmyristylated equine infectious anemia virus (EIAV) MA and its interaction with PIP2-C4 primarily using solution NMR spectroscopy. The measured 1H-15N residual dipolar coupling agrees with the atomic coordinates from the EIAV MA crystal structure. The analytical ultracentrifugation results show a dominant population of monomeric EIAV MA at a concentration of 63 microM and 20 degrees C, along with a small trimer and a broad distribution of other oligomers. The monomer-trimer equilibrium model and the quaternary packing of the trimer were further established by the concentration-dependent 15N spin relaxation rates and chemical shifts. Binding of MA to PIP2-C4 was detected by chemical shift mapping (CSM) with an apparent Kd of 182 +/- 56 microM, a value similar to that reported for HIV-1 MA. The PIP2 binding site includes the Loop region between Helix2 and Helix3 in the EIAV MA. CSM and spin relaxation dispersion reveal a coupling of conformational change and submillisecond dynamics, respectively, between the Loop and trimeric Interface Residues due to PIP2 binding. We infer that PIP2 participates in the initial trimer formation of EIAV MA, but more importantly, the concentration effect is dominant in shifting the equilibrium toward trimer, in line with the entropic switch mechanism proposed for myristylated HIV-1 MA.