Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate.

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Prospects for developing an Hepatitis C virus E1E2-based nanoparticle vaccine.

Globally, more than 58 million people are chronically infected with Hepatitis C virus (HCV) with 1.5 million new infections occurring each year. An effective vaccine for HCV is therefore a major unmet medical and public health need. Since HCV rapidly accumulates mutations, vaccines must elicit the production of broadly neutralising antibodies (bnAbs) in a reproducible fashion. Decades of research have generated a number of HCV vaccine candidates. Based on the available data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice, but robust induction of humoral and cellular responses leading to virus neutralisation has not yet been achieved. One issue that has arisen in developing an HCV vaccine (and many other vaccines as well) is the platform used for antigen delivery. The majority of viral vaccine trials have employed subunit vaccines. However, subunit vaccines often have limited immunogenicity, as seen for HCV, and thus multiple formats must be examined in order to elicit a robust anti-HCV immune response. Nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. This review discusses the potential for development of a nanoparticle-based HCV E1E2 vaccine, with an emphasis on the potential benefits of such an approach along with the major challenges facing the incorporation of E1E2 into nanoparticulate delivery systems and how those challenges can be addressed.

Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Structure-Based Design of Hepatitis C Virus E2 Glycoprotein Improves Serum Binding and Cross-Neutralization.

An effective vaccine for hepatitis C virus (HCV) is a major unmet need, and it requires an antigen that elicits immune responses to key conserved epitopes. Based on structures of antibodies targeting HCV envelope glycoprotein E2, we designed immunogens to modulate the structure and dynamics of E2 and favor induction of broadly neutralizing antibodies (bNAbs) in the context of a vaccine. These designs include a point mutation in a key conserved antigenic site to stabilize its conformation, as well as redesigns of an immunogenic region to add a new N-glycosylation site and mask it from antibody binding. Designs were experimentally characterized for binding to a panel of human monoclonal antibodies (HMAbs) and the coreceptor CD81 to confirm preservation of epitope structure and preferred antigenicity profile. Selected E2 designs were tested for immunogenicity in mice, with and without hypervariable region 1, which is an immunogenic region associated with viral escape. One of these designs showed improvement in polyclonal immune serum binding to HCV pseudoparticles and neutralization of isolates associated with antibody resistance. These results indicate that antigen optimization through structure-based design of the envelope glycoproteins is a promising route to an effective vaccine for HCV.IMPORTANCE Hepatitis C virus infects approximately 1% of the world's population, and no vaccine is currently available. Due to the high variability of HCV and its ability to actively escape the immune response, a goal of HCV vaccine design is to induce neutralizing antibodies that target conserved epitopes. Here, we performed structure-based design of several epitopes of the HCV E2 envelope glycoprotein to engineer its antigenic properties. Designs were tested in vitro and in vivo, demonstrating alteration of the E2 antigenic profile in several cases, and one design led to improvement of cross-neutralization of heterologous viruses. This represents a proof of concept that rational engineering of HCV envelope glycoproteins can be used to modulate E2 antigenicity and optimize a vaccine for this challenging viral target.

A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone.

BACKGROUND: Multimeric naphthoquinones are redox-active compounds that exhibit antineoplastic, antiprotozoal, and antiviral activities. Due to their multimodal effect on perturbation of cellular oxidative state, these compounds hold great potential as therapeutic agents against highly proliferative neoplastic cells. In our previous work, we developed a series of novel dimeric naphthoquinones and showed that they were selectively cytotoxic to human acute myeloid leukemia (AML), breast and prostate cancer cell lines. We subsequently identified the oxidoreductase NAD(P)H dehydrogenase, quinone 1 (NQO1) as the major target of dimeric naphthoquinones and proposed a mechanism of action that entailed induction of a futile redox cycling. RESULTS: Here, for the first time, we describe a direct physical interaction between the bromohydroxy dimeric naphthoquinone E6a and NQO1. Moreover, our studies reveal an extensive binding interface between E6a and the isoalloxazine ring of the flavin adenine dinucleotide (FAD) cofactor of NQO1 in addition to interactions with protein side chains in the active site. We also present biochemical evidence that dimeric naphthoquinones affect the redox state of the FAD cofactor of NQO1. Comparison of the mode of binding of E6a with those of other chemotherapeutics reveals unique characteristics of the interaction that can be leveraged in future drug optimization efforts. CONCLUSION: The first structure of a dimeric naphthoquinone-NQO1 complex was reported, which can be used for design and synthesis of more potent next generation dimeric naphthoquinones to target NQO1 with higher affinity and specificity.

Covalent small molecule inhibitors of Ca(2+)-bound S100B.

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca(2+)-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNA(S100B)) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B-p53 complex and restore active p53 in this deadly cancer. Using a structure-activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1-3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX-S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an S100B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity.

The crystal structure of human quinolinic acid phosphoribosyltransferase in complex with its inhibitor phthalic acid.

Quinolinic acid (QA), a biologically potent but neurodestructive metabolite is catabolized by quinolinic acid phosphoribosyltransferase (QPRT) in the first step of the de novo NAD(+) biosynthesis pathway. This puts QPRT at the junction of two different pathways, that is, de novo NAD(+) biosynthesis and the kynurenine pathway of tryptophan degradation. Thus, QPRT is an important enzyme in terms of its biological impact and its potential as a therapeutic target. Here, we report the crystal structure of human QPRT bound to its inhibitor phthalic acid (PHT) and kinetic analysis of PHT inhibition of human QPRT. This structure, determined at 2.55 Å resolution, shows an elaborate hydrogen bonding network that helps in recognition of PHT and consequently its substrate QA. In addition to this hydrogen bonding network, we observe extensive van der Waals contacts with the PHT ring that might be important for correctly orientating the substrate QA during catalysis. Moreover, our crystal form allows us to observe an intact hexamer in both the apo- and PHT-bound forms in the same crystal system, which provides a direct comparison of unique subunit interfaces formed in hexameric human QPRT. We call these interfaces "nondimeric interfaces" to distinguish them from the typical dimeric interfaces observed in all QPRTs. We observe significant changes in the nondimeric interfaces in the QPRT hexamer upon binding PHT. Thus, the new structural and functional features of this enzyme we describe here will aid in understanding the function of hexameric QPRTs, which includes all eukaryotic and select prokaryotic QPRTs.

Hepatitis C Virus E1E2 Structure, Diversity, and Implications for Vaccine Development.

Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.

Site-directed neutralizing antibodies targeting structural sites on SARS-CoV-2 spike protein.

'Epivolve' (epitope evolution) is an innovative paratope-evolving technology using a haptenated peptide or protein immunogen as a means of directing the in vivo immune response to specifically targeted sites at a one amino acid residue resolution. Guided by protein structural analysis, Epivolve technology was tested to develop site-directed neutralizing antibodies (nAbs) in a systematic fashion against the SARS-CoV-2 Receptor Binding Domain (RBD). Thirteen solvent-exposed sites covering the ACE2 receptor-binding interface were targeted. Immunogens composed of each targeted site were used to immunize rabbits in separate cohorts. In vivo site-directed immune responses against all 13 targets were demonstrated by B cell secreted IgG and recombinant IgG testing. One site, SL13 (Y505) which mutates from tyrosine to histidine in the SARS-CoV-2 Omicron variant, was chosen as a proof-of-concept (PoC) model for further functional monoclonal antibody development. Epivolve technology demonstrated the capabilities of generating pan-variant antibodies and nAbs against the SARS-CoV-2 primary strain and the Omicron variant.

Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein.

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

Structural and functional analysis of the pro-domain of human cathelicidin, LL-37.

Cathelicidins form a family of small host defense peptides distinct from another class of cationic antimicrobial peptides, the defensins. They are expressed as large precursor molecules with a highly conserved pro-domain known as the cathelin-like domain (CLD). CLDs have high degrees of sequence homology to cathelin, a protein isolated from pig leukocytes and belonging to the cystatin family of cysteine protease inhibitors. In this report, we describe for the first time the X-ray crystal structure of the human CLD (hCLD) of the sole human cathelicidin, LL-37. The structure of the hCLD, determined at 1.93 Å resolution, shows the cystatin-like fold and is highly similar to the structure of the CLD of the pig cathelicidin, protegrin-3. We assayed the in vitro antibacterial activities of the hCLD, LL-37, and the precursor form, pro-cathelicidin (also known as hCAP18), and we found that the unprocessed protein inhibited the growth of Gram-negative bacteria with efficiencies comparable to that of the mature peptide, LL-37. In addition, the antibacterial activity of LL-37 was not inhibited by the hCLD intermolecularly, because exogenously added hCLD had no effect on the bactericidal activity of the mature peptide. The hCLD itself lacked antimicrobial function and did not inhibit the cysteine protease, cathepsin L. Our results contrast with previous reports of hCLD activity. A comparative structural analysis between the hCLD and the cysteine protease inhibitor stefin A showed why the hCLD is unable to function as an inhibitor of cysteine proteases. In this respect, the cystatin scaffold represents an ancestral structural platform from which proteins evolved divergently, with some losing inhibitory functions.

Structure of human apurinic/apyrimidinic endonuclease 1 with the essential Mg2+ cofactor.

Apurinic/apyrimidinic endonuclease 1 (APE1) mediates the repair of abasic sites and other DNA lesions and is essential for base-excision repair and strand-break repair pathways. APE1 hydrolyzes the phosphodiester bond at abasic sites, producing 5'-deoxyribose phosphate and the 3'-OH primer needed for repair synthesis. It also has additional repair activities, including the removal of 3'-blocking groups. APE1 is a powerful enzyme that absolutely requires Mg2+, but the stoichiometry and catalytic function of the divalent cation remain unresolved for APE1 and for other enzymes in the DNase I superfamily. Previously reported structures of DNA-free APE1 contained either Sm3+ or Pb2+ in the active site. However, these are poor surrogates for Mg2+ because Sm3+ is not a cofactor and Pb2+ inhibits APE1, and their coordination geometry is expected to differ from that of Mg2+. A crystal structure of human APE1 was solved at 1.92 Šresolution with a single Mg2+ ion in the active site. The structure reveals ideal octahedral coordination of Mg2+ via two carboxylate groups and four water molecules. One residue that coordinates Mg2+ directly and two that bind inner-sphere water molecules are strictly conserved in the DNase I superfamily. This structure, together with a recent structure of the enzyme-product complex, inform on the stoichiometry and the role of Mg2+ in APE1-catalyzed reactions.

Glycosylation shapes the efficacy and safety of diverse protein, gene and cell therapies.

Over recent decades, therapeutic proteins have had widespread success in treating a myriad of diseases. Glycosylation, a near universal feature of this class of drugs, is a critical quality attribute that significantly influences the physical properties, safety profile and biological activity of therapeutic proteins. Optimizing protein glycosylation, therefore, offers an important avenue to developing more efficacious therapies. In this review, we discuss specific examples of how variations in glycan structure and glycoengineering impacts the stability, safety, and clinical efficacy of protein-based drugs that are already in the market as well as those that are still in preclinical development. We also highlight the impact of glycosylation on next generation biologics such as T cell-based cancer therapy and gene therapy.

Structure of engineered hepatitis C virus E1E2 ectodomain in complex with neutralizing antibodies.

Hepatitis C virus (HCV) is a major global health burden as the leading causative agent of chronic liver disease and hepatocellular carcinoma. While the main antigenic target for HCV-neutralizing antibodies is the membrane-associated E1E2 surface glycoprotein, the development of effective vaccines has been hindered by complications in the biochemical preparation of soluble E1E2 ectodomains. Here, we present a cryo-EM structure of an engineered, secreted E1E2 ectodomain of genotype 1b in complex with neutralizing antibodies AR4A, HEPC74, and IGH520. Structural characterization of the E1 subunit and C-terminal regions of E2 reveal an overall architecture of E1E2 that concurs with that observed for non-engineered full-length E1E2. Analysis of the AR4A epitope within a region of E2 that bridges between the E2 core and E1 defines the structural basis for its broad neutralization. Our study presents the structure of an E1E2 complex liberated from membrane via a designed scaffold, one that maintains all essential structural features of native E1E2. The study advances the understanding of the E1E2 heterodimer structure, crucial for the rational design of secreted E1E2 antigens in vaccine development.

Skin Vaccination with Ebola Virus Glycoprotein Using a Polyphosphazene-Based Microneedle Patch Protects Mice against Lethal Challenge.

Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (poly[di(carboxylatophenoxy)phosphazene], PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.

Alternatively expressed domains of AU-rich element RNA-binding protein 1 (AUF1) regulate RNA-binding affinity, RNA-induced protein oligomerization, and the local conformation of bound RNA ligands.

AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3'-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.

Structural and Biophysical Characterization of the HCV E1E2 Heterodimer for Vaccine Development.

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.

Engineering subtilisin proteases that specifically degrade active RAS.

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Unique properties of the Mtr4p-poly(A) complex suggest a role in substrate targeting.

Mtr4p is a DEVH-box helicase required for 3'-end processing and degradation of various nuclear RNA substrates. In particular, Mtr4p is essential for the creation of 5.8S rRNA, U4 snRNA, and some snoRNAs and for the degradation of cryptic unstable transcripts (CUTs), aberrant mRNAs, and aberrant tRNAs. Many instances of 3'-end processing require limited polyadenylation to proceed. While polyadenylation can signal degradation in species from bacteria to humans, the mechanism whereby polyadenylated substrates are delivered to the degradation machinery is unknown. Our previous work has shown that Mtr4p preferentially binds poly(A) RNA. We suspect that this preference aids in targeting polyadenylated RNAs to the exosome. In these studies, we have investigated the mechanism underlying the preference of Mtr4p for poly(A) substrates as a means of understanding how Mtr4p might facilitate targeting. Our analysis has revealed that recognition of poly(A) substrates involves sequence-specific changes in the architecture of Mtr4p-RNA complexes. Furthermore, these differences significantly affect downstream activities. In particular, homopolymeric stretches like poly(A) ineffectively stimulate the ATPase activity of Mtr4p and suppress the rate of dissociation of the Mtr4p-RNA complex. These findings indicate that the Mtr4p-poly(A) complex is unique and ideally suited for targeting key substrates to the exosome.

Contributions of the histidine side chain and the N-terminal alpha-amino group to the binding thermodynamics of oligopeptides to nucleic acids as a function of pH.

Interactions of histidine with nucleic acid phosphates and histidine pK(a) shifts make important contributions to many protein-nucleic acid binding processes. To characterize these phenomena in simplified systems, we quantified binding of a histidine-containing model peptide HWKK ((+)NH(3)-His-Trp-Lys-Lys-NH(2)) and its lysine analogue KWKK ((+)NH(3)-Lys-Trp-Lys-Lys-NH(2)) to a single-stranded RNA model, polyuridylate (polyU), by changes in tryptophan fluorescence as a function of salt concentration and pH. For both HWKK and KWKK, equilibrium binding constants, K(obs), and magnitudes of log-log salt derivatives, SK(obs) identical with (partial differential logK(obs)/partial differential log[Na(+)]), decreased with increasing pH in the manner expected for a titration curve model in which deprotonation of the histidine and alpha-amino groups weakens binding and reduces its salt-dependence. Fully protonated HWKK and KWKK exhibit the same K(obs) and SK(obs) within uncertainty, and these SK(obs) values are consistent with limiting-law polyelectrolyte theory for +4 cationic oligopeptides binding to single-stranded nucleic acids. The pH-dependence of HWKK binding to polyU provides no evidence for pK(a) shifts nor any requirement for histidine protonation, in stark contrast to the thermodynamics of coupled protonation often seen for these cationic residues in the context of native protein structure where histidine protonation satisfies specific interactions (e.g., salt-bridge formation) within highly complementary binding interfaces. The absence of pK(a) shifts in our studies indicates that additional Coulombic interactions across the nonspecific-binding interface between RNA and protonated histidine or the alpha-amino group are not sufficient to promote proton uptake for these oligopeptides. We present our findings in the context of hydration models for specific vs nonspecific nucleic acid binding.