Search

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

KAT6A is a lysine histone acetyltransferase (HAT) of the MYST family of HATs. KAT6A, and its paralog KAT6B, have been shown to acetylate histone H3K23Ac and regulate diverse biological processes, including transcription, cell-cycle progression, stem cell maintenance and development. Molecular dysregulation of KAT6A has been observed in several cancers, including amplifications in breast, lung, ovarian cancer along with oncogenic fusions in AML. In breast cancer, KAT6A is amplified as part of the 8p11 amplicon in 10-15% of the patient population, which correlates with a worse clinical outcome in the estrogen receptor+ (ER+) subtype. Here we present identification of a first-in-class potent KAT6A/KAT6B tool inhibitor CTx-648 (PF-9363), that possesses high selectivity versus other MYST family members (KAT7, KAT5, KAT8) and other KATs, demonstrating anti-tumor activity in breast cancer. Using genetic and pharmacological approaches, we have demonstrated several ER+ breast cancer cell lines including KAT6A amplified and over-expressing models, are dependent on KAT6A enzymatic function. Epigenomic profiling studies using bulk and nascent RNA-seq combined with ATAC-seq revealed CTx-648 leads to downregulation of a specific set of genes involved in ESR1 pathway, cell cycle and stem cell pathways. In vivo target validation studies showed strong anti-tumor activity of CTx-648 in several ER+ breast cancer cell line and patient-derived xenograft models, including models harboring endocrine therapy resistance ESR1 mutations, highlighting promise for this novel therapy in ER+ breast cancer population. Based on the strength of the pre-clinical data, a selective KAT6 inhibitor (PF-07248144) is now commencing a Phase 1 clinical study in Advanced or Metastatic Solid Tumors. Citation Format: Shikhar Sharma, Jay Chung, Sean Uryu, Amanda Rickard, Natalie Nady, Showkhin Khan, Zhenxiong Wang, Yong Zhang, Haikuo Zhang, Pei-Pei Kung, Eric Greenwald, Karen Maegley, Patrick Bingham, Hieu Lam, Ylva E. Bozikis, Hendrik Falk, Elizabeth Allan, Vicky M. Avery, Miriam S. Butler, Michelle A. Camerino, Catalina Carrasco-Pozo, Susan A. Charman, Melissa J. Davis, Mark A. Dawson, Dawson Sarah-Jane, Melanie de Silva, Matthew L. Dennis, Olan Dolezal, Rachel Lagiakos, Geoffrey J. Lindeman, Laura MacPherson, Stewart Nuttall, Thomas S. Peat, Bin Ren, Alexandra E. Stupple, Elliot Surgenor, Chin Wee Tan, Tim Thomas, Jane E. Visvader, Anne K. Voss, Francois Vaillant, Karen L. White, James Whittle, Yuqing Yang, Soroor Hediyeh-Zadeh, Paul A. Stupple, Ian P. Street, Brendon J. Monahan, Thomas Paul. First-in-class KAT6A/KAT6B inhibitor CTx-648 (PF-9363) demonstrates potent anti-tumor activity in ER+ breast cancer with KAT6A dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1130.

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor.

The WD40-repeat protein WDR5 scaffolds various epigenetic writers and is a critical component of the mammalian SET/MLL histone methyltransferase complex. Dysregulation of the MLL1 catalytic function is associated with mixed-lineage leukemia, and antagonism of the WDR5-MLL1 interaction by small molecules has been proposed as a therapeutic strategy for MLL-rearranged cancers. Small molecule binders of the “WIN” site of WDR5 that cause displacement from chromatin have been additionally implicated to be of broader use in cancer treatment. In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-containing compound that is bound in the WIN site. The subsequent medicinal chemistry campaign—guided by a suite of high-resolution cocrystal structures with WDR5—progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine; a tripeptide containing one such substitution was resolved in a high resolution structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (kd = ∼0.06 s−1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics.

Gram-positive bacteria utilise class A sortases to coat the surface of their cells with a diversity of proteins that facilitate interactions with their environment and play fundamental roles in cell physiology and virulence. A putative sortase A gene was identified in the genome of the poorly studied meat spoilage bacterium Brochothrix thermosphacta. To understand how this bacterium mediates interactions with its environment, an N-terminal truncated, His-tagged variant of this protein (His6-BtSrtA) was expressed and purified. Catalytic activity of recombinant His6-BtSrtA was investigated, including sorting motif recognition of target proteins and bioconjugation activity. Further, the B. thermosphacta genome was examined for the presence of sortase A (SrtA) protein substrates. His6-BtSrtA readily formed intermediate complexes with LPXTG-tagged proteins. Although the reaction was inefficient, nucleophilic attack of the resultant thioacyl intermediates by tri-glycine was observed. Genome examination identified 11 potential SrtA substrates, two of which contained protein domains associated with adherence of pathogens to host extracellular matrix proteins and cells, suggesting the B. thermosphacta SrtA may be indirectly involved in its attachment to meat surfaces. Thus, further work in this area could provide crucial insight into molecular mechanisms involved in the colonisation of meat by B. thermosphacta.

Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA) and a ~32 kDa subunit B (RTB). There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs), previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR) array biosensor (ProteOn XPR36) indicated three distinct, non-competitive binding epitopes (“bins”). The association (k a ) and dissociation (k d ) rate constants and binding affinities (K D ) of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (K D ) ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab) fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications.

The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal.

The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (V(NAR)) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. V(NAR)s possess advantages in stability, size (~14 kDa) and cryptic epitope recognition compared to conventional antibodies. The V(NAR) domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the V(NAR) was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.

One method of laboratory- or field-based testing for anthrax is detection of Bacillus anthracis spores by high-affinity, high specificity binding reagents. From a pool of monoclonal antibodies, we selected one such candidate (A4D11) with high affinity for tBclA, a truncated version of the B. anthracis exosporium protein BclA. Kinetic analysis utilising both standard and kinetic titration on a Biacore biosensor indicated antibody affinities in the 300 pM range for recombinant tBclA, and the A4D11 antibody was also re-formatted into scFv configuration with no loss of affinity. However, assays against B. anthracis and related Bacilli species showed limited binding of intact spores as well as significant cross-reactivity between species. These results were rationalized by determination of the three-dimensional crystallographic structure of the scFv-tBclA complex. A4D11 binds the side of the tBclA trimer, contacting a face of the antigen normally packed against adjacent trimers within the exosporium structure; this inter-spore interface is highly conserved between Bacilli species. Our results indicate the difficulty of generating a high-affinity antibody to differentiate between the highly conserved spore structures of closely related species, but suggest the possibility of future structure-based antibody design for this difficult target. Proteins 2011. © 2011 Wiley-Liss, Inc.

Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark V(NAR) single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors.

Summary Apical membrane antigen 1 (AMA1) is essential for invasion of erythrocytes and hepatocytes by Plasmodium parasites and is a leading malarial vaccine candidate. Although conventional antibodies to AMA1 can prevent such invasion, extensive polymorphisms within surface-exposed loops may limit the ability of these AMA1-induced antibodies to protect against all parasite genotypes. Using an AMA1-specific IgNAR single-variable-domain antibody, we performed targeted mutagenesis and selection against AMA1 from three P. falciparum strains. We present cocrystal structures of two antibody-AMA1 complexes which reveal extended IgNAR CDR3 loops penetrating deep into a hydrophobic cleft on the antigen surface and contacting residues conserved across parasite species. Comparison of a series of affinity-enhancing mutations allowed dissection of their relative contributions to binding kinetics and correlation with inhibition of erythrocyte invasion. These findings provide insights into mechanisms of single-domain antibody binding, and may enable design of reagents targeting otherwise cryptic epitopes in pathogen antigens.

The structure of C. perfringens sortase D was determined at 1.99 Å resolution. Comparative biochemical and structural analyses revealed that this transpeptidase may represent a new subclass of the sortase D family., The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.

Protein scaffolds derived from non-immunoglobulin sources are increasingly being adapted and engineered to provide unique binding molecules with a diverse range of targeting specificities. The ColE7 immunity protein (Im7) from Escherichia coli is potentially one such molecule, as it combines the advantages of (i) small size, (ii) stability conferred by a conserved four anti-parallel alpha-helical framework and (iii) availability of variable surface loops evolved to inactivate members of the DNase family of bacterial toxins, forming one of the tightest known protein-protein interactions. Here we describe initial cloning and protein expression of Im7 and its cognate partner the 15 kDa DNase domain of the colicin E7. Both proteins were produced efficiently in E.coli, and their in vitro binding interactions were validated using ELISA and biosensor. In order to assess the capacity of the Im7 protein to accommodate extensive loop region modifications, we performed extensive molecular modelling and constructed a series of loop graft variants, based on transfer of the extended CDR3 loop from the IgG1b12 antibody, which targets the gp120 antigen from HIV-1. Loop grafting in various configurations resulted in chimeric proteins exhibiting retention of the underlying framework conformation, as measured using far-UV circular dichroism spectroscopy. Importantly, there was low but measurable transfer of antigen-specific affinity. Finally, to validate Im7 as a selectable scaffold for the generation of molecular libraries, we displayed Im7 as a gene 3 fusion protein on the surface of fd bacteriophages, the most common library display format. The fusion was successfully detected using an anti-Im7 rabbit polyclonal antibody, and the recombinant phage specifically recognized the immobilized DNase. Thus, Im7 scaffold is an ideal protein display scaffold for the future generation and for the selection of libraries of novel binding proteins.

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an “upright” conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface.

The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2- and 2.8-Å structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the “bottom” of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain/variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended β-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

Haloviruses HF1 and HF2 were isolated from the same saltern pond and are adapted to hypersaline conditions, where they infect a broad range of haloarchaeal species. The HF2 genome has previously been reported. The complete sequence of the HF1 genome has now been determined, mainly by PCR and primer walking. It was 75,898 bp in length and was 94.4% identical to the HF2 genome but about 1.8 kb shorter. A total of 117 open reading frames and five tRNA-like genes were predicted, and their database matches and characteristics were similar to those found in HF2. A comparison of the predicted restriction digest patterns based on nucleotide sequence with the observed restriction digest patterns of viral DNA showed that, unlike the case for HF2, some packaged HF1 DNA had cohesive termini. Except for a single base change, HF1 and HF2 were identical in sequence over the first 48 kb, a region that includes the early and middle genes. The remaining 28 kb of HF1 showed many differences from HF2, and the similarity of the two genomes over this late gene region was 87%. The abrupt shift in sequence similarity around 48 kb suggests a recent recombination event between either HF1 or HF2 and another HF-like halovirus that has swapped most of the right-end 28 kb. This example indicates there is a high level of recombination among viruses that live in this extreme environment.

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

HF2 is a haloarchaeal virus infecting two Halorubrum species (Family Halobacteriaceae). It is lytic, has a head-and-tail morphology and belongs to the Myoviridae (contractile tails). The linear double-stranded DNA genome was sequenced and found to be 77 670 bp in length, with a mol% G+C of 55.8. A total of 121 likely open reading frames (ORFs) were identified, of which 37 overlapped at start and stop codons. The predicted proteins were usually acidic (average pI of 4.8), and less than about 12% of them had homologues in the sequence databases. Four complete tRNA-like sequences (tRNA-Arg, -Asx, -Pro and -Tyr) and an incomplete tRNA-Thr were detected. A transcription map showed that most of the genome was transcribed and that the synthesis of transcripts occurred in a highly organized and reproducible pattern over a 5 h infection cycle. Transcripts often spanned multiple ORFs, suggesting that viral genes were organized into operons. The predicted ORF and observed transcript directions matched well and showed that transcription is mainly directed inwards from the genome termini, meeting at about 45-48 kb, and this was also a turning point in a cumulative GC-skew plot. The low point in cumulative GC-skew, near the left end, was a region rich in short repeats and lacking ORFs, which is likely to be an origin of replication. The HF2 genome is a mosaic of components from widely different sources, demonstrating clearly that viruses of haloarchaea, like their bacteriophage counterparts, are vectors for the exchange and transmission of genetic material between wide taxonomic distances, even across domains.

The new antigen receptor (NAR) from sharks consists of a single immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antibody. Two closely related NARs with affinity for the Kgp (lysine-specific) gingipain protease from Porphyromonas gingivalis were selected by panning an NAR variable domain library. When produced in Escherichia coli, these recombinant NARs were stable, correctly folded, and specifically bound Kgp (Kd=1.31±0.26×10−7 M). Binding localised to the Kgp adhesin domains, however without inhibiting adhesin activity. These naturally occurring proteins indicate an immune response to pathogenic bacteria and suggest that the NAR is a true antibody-like molecule.

Self association of the amyloid-β (Aβ42) peptide into oligomers, high molecular weight forms, fibrils and ultimately neuritic plaques, has been correlated with progressive cognitive decline in Alzheimer’s disease. Thus, insights into the drivers of the aggregation pathway have the capacity to significantly contribute to our understanding of disease mechanism. Functional assays and a three-dimensional crystal structure of the P3 amyloidogenic region 18–41 of Aβ were used to identify residues important in self-association and to design novel non-aggregating variants of the peptide. Biophysical studies (gel filtration, SDS–PAGE, dynamic light scattering, thioflavin T assay, and electron microscopy) demonstrate that in contrast to wild type Aβ these targeted mutations lose the ability to self-associate. Loss of aggregation also correlates with reduced neuronal toxicity. Our results highlight residues and regions of the Aβ peptide important for future targeting agents aimed at the amelioration of Alzheimer’s disease.

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

We have designed a gene cassette for expression of the bleomycin-resistance protein from Streptoalloteichus hindustanus (ShBle) in the extremely halophilic archaeon Haloferax volcanii, and shown that transformed haloarchaea are resistant to bleomycin. Recombinant ShBle was purified by a one-step affinity-chromatography procedure as a correctly folded, dimeric protein. ShBle thus provides a useful haloarchaeal selectable marker and represents the first non-halophilic and soluble heterologous protein to be expressed in the Haloarchaea.

We have selected the Streptoalloteichus hindustanus bleomycin-resistance protein ShBle, a 28-kDa homodimer, as a scaffold for the display of bioactive peptides and other peptide epitopes. To create a monomeric scaffold, we investigated the effect of mutating residue proline 9 to glycine. This residue plays a critical role in ShBle dimerization by affecting the position of the eight N-terminal residues which secure the interaction between the monomeric subunits. We demonstrate that this mutation weakens the dimerization interaction, resulting in establishment of a stable equilibrium between monomeric and dimeric ShBle species in solution. Circular dichroism and SDS-PAGE data indicate that the Pro9Gly mutation does not disrupt the structure of the molecule. Production of a fully monomeric form of ShBle required complete removal of the eight-residue N-terminal peptide, and the interaction across the now solvent-exposed hydrophobic interface of the ShBle monomer was insufficient to drive dimerization. To demonstrate efficient display of epitope tags on the ShBle protein, we displayed dual-octapeptide FLAG tags at the protein C-terminus. These additions did not interfere with protein folding or activity. The resulting ShBle scaffold was used to compare the efficiency of two commercial FLAG-specific antibodies by biosensor.

Most mitochondrial proteins are nuclear encoded, synthesized on cytosolic ribosomes, and imported into the mitochondria. We have identified and characterized a 309 amino acid human protein with a molecular weight of 34 kDa that functions as a subunit of the translocase for the import of such proteins. hTom34 (34-kDa Translocase of the Outer Mitochondrial Membrane) is displayed on the surface of mitochondria and is resistant to extraction under alkaline conditions. Antibodies raised against hTom34 specifically inhibit in vitro import of the mitochondrial precursor protein preornithine transcarbamylase into mitochondria isolated from rat liver. Based on trypsin digestion experiments, the receptor has a large (27 kDa) C-terminal domain exposed to the cytosol. This novel component of the protein import machinery possesses a 62 residue motif conserved with the Tom70 family of mitochondrial receptors but otherwise appears to have no counterpart so far characterized in the mitochondria of any other species.

Anti-amyloid-β immunotherapies are a promising therapeutic approach for the treatment and prevention of Alzheimer's disease (AD). Single chain antibody fragments (scFv) are an attractive alternative to whole antibodies due to their small size, single polypeptide format and inability to stimulate potentially undesirable Fc-mediated immune effector functions. We have generated the scFv derivative of anti-Aβ monoclonal antibody, 1E8, known to target residues 17-22 of Aβ. Here we show that the soluble 1E8 scFv binds to the central region of Aβ with an affinity of ~55 nM and significantly reduces fibril formation of Aβ(1-42). Furthermore, 1E8 scFv ameliorates Aβ(1-42)-mediated toxicity in the PC12 cell line and murine primary neuronal cultures. This ability to both target the central region of Aβ and prevent Aβ(1-42) neurotoxicity in vitro makes it a promising therapeutic antibody building block for further functionalization, toward the treatment of AD.

The single variable new antigen receptor domain antibody fragments (VNARs) derived from shark immunoglobulin new antigen receptor antibodies (IgNARs) represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1) from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs) in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

Immunoglobulin new antigen receptors (IgNARs) from sharks are a distinct class of immune receptors, consisting of homodimers with no associated light chains. Antigen binding is encapsulated within single VNAR immunoglobulin domains of 13-14 kDa in size. This small size and single domain format means that they exhibit considerable stability and are readily produced in heterologous protein expression systems. In this chapter, I describe the history and discovery of IgNARs, the development of VNAR biotechnology, and highlight important factors in VNAR protein production.

Immunoglobulin new antigen receptors (IgNARs) from sharks are a distinct class of immune receptors, consisting of homodimers with no associated light chains. Antigen binding is encapsulated within single VNAR immunoglobulin domains of 13-14 kDa in size. This small size and single domain format means that they exhibit considerable stability and are readily produced in heterologous protein expression systems. In this chapter, I describe the history and discovery of IgNARs, the development of VNAR biotechnology, and highlight important factors in VNAR protein production.

Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction.

Alzheimer's disease is a progressive neurodegenerative disorder associated with the presence of amyloid-β (Aβ) peptide fibrillar plaques in the brain. However, current evidence suggests that soluble nonfibrillar Aβ oligomers may be the major drivers of Aβ-mediated synaptic dysfunction. Structural information on these Aβ species has been very limited because of their noncrystalline and unstable nature. Here, we describe a crystal structure of amylogenic residues 18–41 of the Aβ peptide (equivalent to the p3 α/γ-secretase fragment of amyloid precursor protein) presented within the CDR3 loop region of a shark Ig new antigen receptor (IgNAR) single variable domain antibody. The predominant oligomeric species is a tightly associated Aβ dimer, with paired dimers forming a tetramer in the crystal caged within four IgNAR domains, preventing uncontrolled amyloid formation. Our structure correlates with independently observed features of small nonfibrillar Aβ oligomers and reveals conserved elements consistent with residues and motifs predicted as critical in Aβ folding and oligomerization, thus potentially providing a model system for nonfibrillar oligomer formation in Alzheimer's disease.

Two novel halophilic archaebacterial bacteriophages, HF1 and HF2, were isolated from an Australian solar saltern. They were morphologically identical with icosahedral-shaped heads (diameter 58 nm) and contractile tails (length 94 nm). Other similarities included sensitivity to reduced ionic conditions, similar protein profiles by SDS-PAGE, and dsDNA genomes of identical size (73.5 kbp) with analogous restriction patterns. DNA-DNA hybridization data showed the two phages to be closely related. HF1 has a broad host-range, infecting members of three halobacterial genera including Halobacterium salinarium and the genetically well-characterized strain Haloferax volcanii WFD11. Mutants showing increased plating efficiency on alternative hosts were readily selectable. By contrast, HF2 showed a limited host range, confined to the closely related dam-methylated strains Ch2 and H. saccharovorum.

One method of laboratory- or field-based testing for anthrax is detection of Bacillus anthracis spores by high-affinity, high specificity binding reagents. From a pool of monoclonal antibodies, we selected one such candidate (A4D11) with high affinity for tBclA, a truncated version of the B. anthracis exosporium protein BclA. Kinetic analysis utilising both standard and kinetic titration on a Biacore biosensor indicated antibody affinities in the 300 pM range for recombinant tBclA, and the A4D11 antibody was also re-formatted into scFv configuration with no loss of affinity. However, assays against B. anthracis and related Bacilli species showed limited binding of intact spores as well as significant cross-reactivity between species. These results were rationalized by determination of the three-dimensional crystallographic structure of the scFv-tBclA complex. A4D11 binds the side of the tBclA trimer, contacting a face of the antigen normally packed against adjacent trimers within the exosporium structure; this inter-spore interface is highly conserved between Bacilli species. Our results indicate the difficulty of generating a high-affinity antibody to differentiate between the highly conserved spore structures of closely related species, but suggest the possibility of future structure-based antibody design for this difficult target.

We report here on advances made in the construction of plasmid shuttle vectors suitable for genetic manipulations in both Escherichia coli and halobacteria. Starting with a 20.4-kb construct, pMDS1, new vectors were engineered which were considerably smaller yet retained several alternative cloning sites. A restriction barrier observed when plasmid DNA was transferred into Haloferax volcanii cells was found to operate via adenine methylation, resulting in a 10(3) drop in transformation efficiency and the loss of most constructs by incorporation of the resistance marker into the chromosome. Passing shuttle vectors through E. coli dam mutants effectively avoided this barrier. Deletion analysis revealed that the gene(s) for autonomous replication of pHK2 (the plasmid endogenous to Haloferax strain Aa2.2 and used in the construction of pMDS1) was located within a 4.2-kb SmaI-KpnI fragment. Convenient restriction sites were identified near the termini of the novobiocin resistance determinant (gyrB), allowing the removal of flanking sequences (including gyrA). These deletions did not appear to significantly affect transformation efficiencies or the novobiocin resistance phenotype of halobacterial transformants. Northern blot hybridization with strand- and gene-specific probes identified a single gyrB-gyrA transcript of 4.7 kb. This is the first demonstration in prokaryotes that the two subunits of DNA gyrase may be cotranscribed.

Protein scaffolds represent a new generation of universal binding frameworks for use as future immunopharmaceuticals to complement the expanding repertoire of therapeutic monoclonal antibodies. Here, we review recent literature describing advances in protein scaffold development, including efforts to engineer the minimal immunoglobulin-based binding-domain and molecular library design. Several diverse protein folds are currently under development on the basis of modular construction, a strategy also observed in families of naturally evolved immune receptors. We describe potential therapeutic and intracellular applications where scaffold-specific features provide distinct advantages for targeting of non-conventional antigens and comment on the scientific progress and validation of several designed scaffolds in the voyage towards first-in-human trials.

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called BclA, which comprises a central collagen-like region (CLR) and a globular C-terminal domain. Here, the entire CLR coding sequence of BclA was removed, and the resulting protein (tBclA) produced in Escherichia coli. The crystallographic structure of tBclA was determined to 1.35 A resolution, and consists of an all-beta structure with a TNF-like jelly fold topology (12 beta-strands which form 2 beta-sheets of five strands each) consistent with previous studies on wild-type BclA. These globular domains are tightly packed into trimeric structures (surface shape complementarity; S (c) = 0.83), demonstrating that formation of the core structure of BclA is independent of the anchoring collagen-like region. A polyclonal antibody raised against tBclA recognized B. anthracis spores directly, and showed little cross-reactivity (

Immunoglobulin new antigen receptors (IgNARs) are unique single domain antibodies found in the serum of sharks. The individual variable (VNAR) domains bind antigen independently and are candidates for the smallest antibody-based immune recognition units (approximately 13 kDa). Here, we first isolated and sequenced the cDNA of a mature IgNAR antibody from the spotted wobbegong shark (Orectolobus maculatus) and confirmed the independent nature of the VNAR domains by dynamic light scattering. Second, we asked which of the reported antibody fragment dimerisation strategies could be applied to VNAR domains to produce small bivalent proteins with high functional affinity (avidity). In contrast to single chain Fv (scFv) fragments, separate IgNARs could not be linked into a tandem single chain format, with the resulting proteins exhibited only monovalent binding due solely to interaction of the N-terminal domain with antigen. Similarly, incorporation of C-terminal helix-turn-helix (dhlx) motifs, while resulting in efficiently dimerised protein, resulted in only a modest enhancement of affinity, probably due to an insufficiently long hinge region linking the antibody to the dhlx motif. Finally, generation of mutants containing half-cystine residues at the VNAR C-terminus produced dimeric recombinant proteins exhibiting high functional affinity for the target antigens, but at the cost of 50-fold decreased protein expression levels. This study demonstrates the potential for construction of bivalent or bispecific IgNAR-based binding reagents of relatively small size (approximately 26 kDa), equivalent to a monovalent antibody Fv fragment, for formulation into future diagnostic and therapeutic formats.

The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.

Marine waters contain virus populations 10–100 times greater than their prokaryote hosts, and, since each host species is probably susceptible to infection by several different viruses, the diversity of these simple parasites is, by any calculation, simply enormous (for a broad review, see Wommack and Colwell 2000). They are a major driving force in host evolution and are likely to play a significant role in moving genes between host lineages (lateral gene transfer). Compared with the more than 4000 known (and relatively easily isolated) bacteriophages, only about 13 haloarchaeal viruses have ever been reported and only a few have been examined in any detail. Even more disappointing is that many of the earlier isolates have probably been lost. Most plaque on strains of Halobacterium salinarum and are specific for that genus (Table 17. 1), but these hosts are notorious for their genetic instability and complex nutrient requirements. For example, in studies of ΦH, the integration of ISH8 was common, leading to virus variants and deletions at high rates (see Reiter et al.1988).

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

Identification of the smallest antibody fragment still capable of binding to antigen has progressed from full antibody molecules to Fab and recombinant single chain Fv fragments. Now, a further reduction to single domain binding proteins based upon immunoglobulin VH and VH-like domains offers exciting prospects in the development of novel immunotherapeutics and immunodiagnostics. Minimisation of the antigen-binding fragment to such small single-domain proteins offers the advantages of enhanced stability, and possibly access to a class of antigenic epitopes not generally recognised by conventional antibodies.

Eukaryotic ribosome complexes can be used as a means to display a library of proteins, and isolate specific binding reagents by screening against target molecules. Here we present, as an example, a method for the display of a library of immunoglobulin variable-like domains (VLDs) for the production of stable mRNA/ribosome/protein complexes. These complexes are produced by the addition of specific in vitro transcriptional promoter elements and translation control sequences to the template DNA. Furthermore, an appropriate spacer (anchor) domain is included for efficient folding of the nascent translated protein, which remains attached to the ribosome complex. Ribosome complexes are panned against hen egg lysozyme-conjugated magnetic beads and genes encoding specific, binding, V-like domains are recovered by RT-PCR and cloned into an Escherichia coli expression vector.

Protein affinity maturation using molecular evolution techniques to produce high-affinity binding proteins is an important step in the generation of reagents for cancer diagnosis and treatment. Currently, the most commonly used molecular evolution processes involve mutation of a single gene into complex gene repertoires followed by selection from a display library. Fd-bacteriophage are the most popular display vectors, but are limited in their capacity for library presentation, speed of processing and mutation frequency. Recently, the potential of ribosome display for directed molecular evolution was recognised and developed into a rapid and simple affinity selection strategy using ribosome complexes to display antibody fragments (scFv). Ribosome display and selection has the potential to generate and display large libraries more representative of the theoretical optima for naive repertoires (10(14)). Even more important is the application of ribosome display for the affinity maturation of individual proteins by rapid mutation and selection cycles. These display strategies can apply to other members of the immunoglobulin superfamily; for example single V-domains which have an important application in providing specific targeting to either novel or refractory cancer markers. We discuss the application of ribosome display and selection in conjunction with variable domain (CTLA-4) libraries as the first step towards this objective and review affinity maturation strategies for in vitro ribosome display systems.

We have characterised a 16.3-kDa human protein that functions as a receptor for the import of preproteins into mitochondria. Based on amino acid sequence alignments, the protein (hMas20p) is 41% similar to Mas20p (20-kDa mitochondrial assembly protein) from yeast Saccharomyces cerevisiae and 38% similar to MOM19 (19-kDa mitochondrial outer-membrane protein) from Neurospora crassa. hMas20p has a putative N-terminal transmembrane sequence of 29 amino acids and an acidic C-terminus. A 13-kDa fragment [des-(1-29)-hMas20p], which lacks the 29-amino acid putative N-terminal transmembrane domain, is soluble when expressed in Escherichia coli. Antibodies produced against this domain crossreacted with a protein of 16 kDa in outer membranes of mitochondria from rat liver and inhibited import of protein into isolated mitochondria from rat liver. In addition, the recombinant soluble domain folds into a functional structure as it competes with hMas20p on the mitochondrial surface for precursor binding, confirming the functional role of hMas20p in the import of preproteins into mitochondria.

A novel halophilic archaeon, strain Ch2, was isolated from a marine solar saltern in Geelong, Australia. The fact that this organism had a dam-methylated genome suggested that it is closely related to the taxon that includes Halobacterium saccharovorum, Halobacterium sodomense, and Halobacterium trapanicum. A sequence analysis of the 16S rRNA gene (Ch2 has three copies of this gene) showed that Ch2 is phylogenetically equidistant from the genera Haloarcula and Haloferax and closely related to H. saccharovorum. The susceptibility of both Ch2 and H. saccharovorum to the recently isolated halophage HF2 supported the hypothesis that these two organisms are closely related.