Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains

We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained against five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.     

Cysteine-containing histone H1-like (PL-I) proteins of sperm.

We have determined the presence of cysteine in the protein PL-I from the sperm of the surf clam Spisula solidissima. The existence of cysteine in this histone H1-related protein is responsible for its previously described aggregation behavior. The location of this residue, within the trypsin-resistant domain of the protein, has been established. We have also shown that cysteine is ubiquitously present in the PL-I proteins from the sperm of other bivalve mollusks but is absent from other PL of smaller molecular mass (PL-II, PL-III, PL-IV). We have also found cysteine to be present in the PL-I from a tunicate (Chelysoma productum) but absent in a PL-I from a fish (Mullus barbatus). The possible significance of the unusual occurrence of cysteine in these histone-H1-related proteins is discussed.     

Distinct polyphosphoinositide binding selectivities for pleckstrin homology domains of GRP1-like proteins based on diglycine versus triglycine motifs

GRP1 and the related proteins ARNO and cytohesin-1 are ARF exchange factors that contain a pleckstrin homology (PH) domain thought to target these proteins to cell membranes through binding polyphosphoinositides. Here we show the PH domains of all three proteins exhibit relatively high affinity for dioctanoyl phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)), with K(D) values of 0.05, 1.6 and 1.0 micrometer for GRP1, ARNO, and cytohesin-1, respectively. However, the GRP1 PH domain was unique among these proteins in its striking selectivity for PtdIns(3,4, 5)P(3) versus phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)), for which it exhibits about 650-fold lower apparent affinity. Addition of a glycine to the Gly(274)-Gly(275) motif in GRP1 greatly increased its binding affinity for PtdIns(4,5)P(2) with little effect on its binding to PtdIns(3,4,5)P(3), while deletion of a single glycine in the corresponding triglycine motif of the ARNO PH domain markedly reduced its binding affinity for PtdIns(4,5)P(2) but not for PtdIns(3,4,5)P(3). In intact cells, the hemagglutinin epitope-tagged PH domain of GRP1 was recruited to ruffles in the cell surface in response to insulin, as were full-length GRP1 and cytohesin-1, but the PH domain of cytohesin-1 was not. These data indicate that the unique diglycine motif in the GRP1 PH domain, as opposed to the triglycine in ARNO and cytohesin-1, directs its remarkable PtdIns(3,4,5)P(3) binding selectivity.     

Perforin evolved from a gene duplication of MPEG1, followed by a complex pattern of gene gain and loss within Euteleostomi

ABSTRACT: BACKGROUND: The pore-forming protein perforin is central to the granule-exocytosis pathway used by cytotoxic lymphocytes to kill abnormal cells. Although this mechanism of killing is conserved in bony vertebrates, cytotoxic cells are present in other chordates and invertebrates, and their cytotoxic mechanism has not been elucidated. In order to understand the evolution of this pathway, here we characterize the origins and evolution of perforin. RESULTS: We identified orthologs and homologs of human perforin in all but one species analysed from Euteleostomi, and present evidence for an earlier ortholog in Gnathostomata but not in more primitive chordates. In placental mammals perforin is a single copy gene, but there are multiple perforin genes in all lineages predating marsupials, except birds. Our comparisons of these many-to-one homologs of human perforin show that they mainly arose from lineagespecific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. We also present evidence that perforin arose from duplication of the ancient MPEG1 gene, and that it shares a common ancestor with the functionally related complement proteins. CONCLUSIONS: The evolution of perforin in vertebrates involved a complex pattern of gene, as well as intron, gain and loss. The primordial perforin gene arose at least 500 million years ago, at around the time that the major histocompatibility complex-T cell receptor antigen recognition system was established. As it is absent from primitive chordates and invertebrates, cytotoxic cells from these lineages must possess a different effector molecule or cytotoxic mechanism.     

Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins

In-depth analysis of protein-protein interaction specificities of the MYB protein family of Arabidopsis thaliana revealed a conserved amino acid signature ([DE]Lx(2)[RK]x(3)Lx(6)Lx(3)R) as the structural basis for interaction between MYB and R/B-like BHLH proteins. The motif has successfully been used to predict new MYB/BHLH interactions for A. thaliana proteins, it allows to discriminate between even closely related MYB proteins and it is conserved amongst higher plants. In A. thaliana, the motif is shared by fourteen R2R3 MYB proteins and six 1R MYB proteins. It is located on helices 1 and 2 of the R3 repeat and forms a characteristic surface-exposed pattern of hydrophobic and charged residues. Single-site mutation of any amino acid of the signature impairs the interaction. Two particular amino acids have been determined to account for most of the interaction stability. Functional specificity of MYB/BHLH complexes was investigated in vivo by a transient DFR promoter activation assay. Residues stabilizing the MYB/BHLH interaction were shown to be critical for promoter activation. By virtue of proved and predicted interaction specificities, this study provides a comprehensive survey of the MYB proteins that interact with R/B-like BHLH proteins potentially involved in the TTG1-dependent regulatory interaction network. The results are discussed with respect to multi-functionality, specificity and redundancy of MYB and BHLH protein function.     

Nep1-like proteins from plant pathogens: recruitment and diversification of the NPP1 domain across taxa

An emerging group of proteins found in many plant pathogens are related to their ability to cause plant cell death. These proteins may be identified by the presence of a common NPP1 (necrosis-inducing Phytophthora protein) domain, and have collectively been named NLPs (Nep1-like proteins). The NLPs are distinguished by their wide distribution across taxa and their broad spectrum of activity against dicotyledonous plants. The function of NLPs is not known but there is strong evidence that they may act as positive virulence factors, accelerating disease and pathogen growth in plant hosts. Interest in NLPs is gaining momentum as more members of this protein family are discovered in more species of plant pathogens.     

Identification of structural domains in proteins by a graph heuristic.

A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward.     

Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.

Two closely related genes coding for NELL proteins (NELL1 and NELL2) have been cloned by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C betaI (PKCbetaI) as bait. The rat NELL proteins show about 55% identity with each other and contain several protein motifs assigned to a secretion signal peptide, an NH(2)-terminal thrombospondin-1 (TSP-1)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains; the NELL proteins share many protein motifs with TSP-1. The NELL proteins expressed in COS-7 cells are homotrimeric glycoproteins and possess heparin-binding activity. Furthermore, while NELL1 and NELL2 show distinct subcellular localization in cytoplasm, they both are partially secreted into the culture medium of COS-7 cells. Although the NELL1 mRNA is faintly expressed in adult neural cells, the NELL2 mRNA is expressed abundantly, particularly in the pyramidal cells of rat hippocampus, showing neuronal high plasticity. During mouse embryogenesis, expression of the NELL2 mRNA is initiated 7-11 days postcoitum, simultaneously with neural plate formation. These results strongly suggest that the NELL2 protein, similar to but not identical with TSP-1, is involved in the growth and differentiation of neural cells. Additionally, the NELL1 and NELL2 mRNAs were found to be expressed abundantly in Burkitt's lymphoma Raji cells and colorectal adenocarcinoma SW480 cells, respectively. Thus, it is likely that the NELL proteins also participate in the growth, differentiation, and oncogenesis of cancer cell lines.     

Biochemical characterization of two Cdc6/ORC1-like proteins from the crenarchaeon Sulfolobus solfataricus

The biological role of archaeal proteins, homologous to the eukaryal replication initiation factors of cell division control (Cdc6) and origin recognition complex (ORC1), has not yet been clearly established. The hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as Sso) possesses three Cdc6/ORC1-like factors, which are named Sso Cdc6-1, Cdc6-2 and Cdc6-3. This study is a report on the biochemical characterization of Sso Cdc6-1 and Cdc6-3. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions by gel filtration analyses. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubble-containing synthetic oligonucleotides. The study has also showed, with surface plasmon resonance measurements, that Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3. These findings may provide important clues needed to understand the biological role that is played by each of these three Cdc6 factors during the DNA replication initiation process in the S. solfataricus cells.     

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.     

Mapping of interaction domains of putative telomere-binding proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana

We previously searched for interactions between plant telomere-binding proteins and found that AtTRB1, from the single-myb-histone (Smh) family, interacts with the Arabidopsis POT1-like-protein, AtPOT1b, involved in telomere capping. Here we identify domains responsible for that interaction. We also map domains in AtTRB1 responsible for interactions with other Smh-family-members. Our results show that the N-terminal OB-fold-domain of AtPOT1b mediates the interaction with AtTRB1. This domain is characteristic for POT1- proteins and is involved with binding the G-rich-strand of telomeric DNA. AtPOT1b also interacts with AtTRB2 and AtTRB3. The central histone-globular-domain of AtTRB1 is involved with binding to AtTRB2 and 3, as well as to AtPOT1b. AtTRB1-heterodimers with other Smh-family-members are more stable than AtTRB1-homodimers. Our results reveal interaction networks of plant telomeres.     

Differential ATP binding and intrinsic ATP hydrolysis by amino-terminal domains of the yeast Mlh1 and Pms1 proteins.

MutL homologs belong to a family of proteins that share a conserved ATP binding site. We demonstrate that amino-terminal domains of the yeast MutL homologs Mlh1 and Pms1 required for DNA mismatch repair both possess independent, intrinsic ATPase activities. Amino acid substitutions in the conserved ATP binding sites concomitantly reduce ATP binding, ATP hydrolysis, and DNA mismatch repair in vivo. The ATPase activities are weak, consistent with the hypothesis that ATP binding is primarily responsible for modulating interactions with other MMR components. Three approaches, ATP hydrolysis assays, limited proteolysis protection, and equilibrium dialysis, provide evidence that the amino-terminal domain of Mlh1 binds ATP with >10-fold higher affinity than does the amino-terminal domain of Pms1. This is consistent with a model wherein ATP may first bind to Mlh1, resulting in events that permit ATP binding to Pms1 and later steps in DNA mismatch repair.     

DNA and histone H1 interact with different domains of HMG 1 and 2 proteins.

High mobility group (HMG) proteins 1 and 2 from calf thymus have been digested under structuring conditions (0.35 M NaCl, pH 7.1) with two proteases of different specificities, trypsin and V8. The two proteases give a different but restricted pattern of peptides in a time course digestion study. However, when the interactions of the peptides with DNA are studied by blotting, a closely related peptide from HMG-1 and -2 does not show any apparent binding. This peptide, from the V8 protease digestion, has been isolated by DNA-cellulose chromatography and has the amino acid composition predicted for a fragment containing the two C-terminal domains of the protein, i.e., approximately residues 74-243 for HMG-1. The same peptide shows the only interaction detectable with labelled histone H1. A separate function for the different domains of HMG proteins 1 and 2 is proposed.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.