Amino Acid Sequence Motifs Research Articles

Molecular characterization of the DNA methyltransferase M1. NcuI from Neisseria cuniculi ATCC 14688

The methyltransferase M1. NcuI is a member of the restriction-modification system in Neisseria cuniculi ATCC14688 and recognizes the asymmetric pentanucleotide sequence 5′-GAAGA-3′/3′-CTTCT-5′. We purified M1. NcuI to electrophoretic homogeneity using a four-step chromatographic procedure. M1. NcuI is a protein with M r = 32,000 ± 1000 under denaturing conditions. It modifies the recognition sequence by transferring the methyl group from S-adenosyl- l-methionine to the 3′ adenine of the pentanucleotide sequence 5′-GAAGA-3′. M1. NcuI, like many other methyltransferases, occurs as a monomer in solution, as determined by gel filtration. Divalent cations inhibit the methylation activity of M1. NcuI. Optimal enzyme activity was observed at a pH of 8.0. M1. NcuI cross-reacted with anti-M1. MboII serum which reflects the similarity of M1. NcuI with M1. MboII at the amino acid level. The gene coding for the enzyme, designated ncuIM1, was cloned, sequenced and overexpressed in Escherichia coli. The structural gene is 780 nucleotides in length coding for a protein of 259 amino acids ( M r 30,098). The presence and distribution of nine highly conserved amino acid sequence motifs and a putative target recognition domain in the enzyme structure suggest that M1. NcuI, similar to M1. MboII and M1. HpyAII, belongs to N 6-adenine β-class DNA methyltransferases.

A second RGD motif in the 1D capsid protein of a SAT1 type foot-and-mouth disease virus field isolate is not essential for attachment to target cells

The amino acid sequence motif Arg-Gly-Asp (RGD), located in the surface-exposed βG-βH loop of the 1D protein of different serotypes and subtypes of foot-and-mouth disease virus (FMDV), is highly conserved and participates in binding of FMDV to susceptible cells. Previous sequence analyses of the 1D-encoding region of a FMDV serotype SAT1 field isolate from Namibia (NAM/307/98) indicated the presence of a second RGD motif upstream of the conserved βG-βH loop RGD. The role of these RGD sequences in virus infection was investigated by mutating the βG-βH loop RGD to a KGE tripeptide, using a genome-length infectious chimeric cDNA clone. Although the infectivity of the derived mutant viruses for baby hamster kidney cells (BHK-21) was lost, subsequent replacement of the KGE sequence with RGD in the mutant cDNA clone led to recovery of infectious viruses. Furthermore, viral RNA replication could be demonstrated with the genetically engineered mutant and non-mutant viruses. The presence of virus particles in the transfected cells could be also demonstrated by electron microscopy. These results demonstrate that, in contrast to the βG-βH loop RGD motif, the second RGD sequence in the capsid protein 1D of NAM/307/98 does not function as a ligand for receptor binding in BHK-21 cells.

The Secreted Protease Factor CPAF Is Responsible for Degrading Pro-apoptotic BH3-only Proteins in Chlamydia trachomatis-infected Cells

The Secreted Protease Factor CPAF Is Responsible for Degrading Pro-apoptotic BH3-only Proteins in Chlamydia trachomatis-infected Cells

Structural Basis and Functional Consequence of Helicobacter pylori CagA Multimerization in Cells

Helicobacter pylori cagA-positive strains are associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells where it localizes to the plasma membrane and undergoes tyrosine phosphorylation at the EPIYA-repeat region, which contains the EPIYA-A segment, EPIYA-B segment, and Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D segment. In host cells, CagA specifically binds to and deregulates SHP-2 phosphatase via the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, thereby inducing an elongated cell shape known as the hummingbird phenotype. In this study, we found that CagA multimerizes in cells in a manner independent of its tyrosine phosphorylation. Using a series of CagA mutants, we identified a conserved amino acid sequence motif (FPLXRXXXVXDLSKVG), which mediates CagA multimerization, within the EPIYA-C segment as well as in a sequence that located immediately downstream of the EPIYA-C or EPIYA-D segment. We also found that a phosphorylation-resistant CagA, which multimerizes but cannot bind SHP-2, inhibits the wild-type CagA-SHP-2 complex formation and abolishes induction of the hummingbird phenotype. Thus, SHP-2 binds to a preformed and tyrosinephosphorylated CagA multimer via its two Src homology 2 domains. These results, in turn, indicate that CagA multimerization is a prerequisite for CagA-SHP-2 interaction and subsequent deregulation of SHP-2. The present work raises the possibility that inhibition of CagA multimerization abolishes pathophysiological activities of CagA that promote gastric carcinogenesis.

Ddi1, a Eukaryotic Protein With the Retroviral Protease Fold

Retroviral aspartyl proteases are homodimeric, whereas eukaryotic aspartyl proteases tend to be large, monomeric enzymes with 2-fold internal symmetry. It has been proposed that contemporary monomeric aspartyl proteases evolved by gene duplication and fusion from a primordial homodimeric enzyme. Recent sequence analyses have suggested that such “fossil” dimeric aspartyl proteases are still encoded in the eukaryotic genome. We present evidence for retention of a dimeric aspartyl protease in eukaryotes. The X-ray crystal structure of a domain of the Saccharomyces cerevisiae protein Ddi1 shows that it is a dimer with a fold similar to that of the retroviral proteases. Furthermore, the double Asp-Thr-Gly-Ala amino acid sequence motif at the active site of HIV protease is found with identical geometry in the Ddi1 structure. However, the putative substrate binding groove is wider in Ddi1 than in the retroviral proteases, suggesting that Ddi1 accommodates bulkier substrates. Ddi1 belongs to a family of proteins known as the ubiquitin receptors, which have in common the ability to bind ubiquitinated substrates and the proteasome. Ubiquitin receptors contain an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain, but Ddi1 is the only representative in which the UBL and UBA domains flank an aspartyl protease-like domain. The remarkable structural similarity between the central domain of Ddi1 and the retroviral proteases, in the global fold and in active-site detail, suggests that Ddi1 functions proteolytically during regulated protein turnover in the cell.

Role of Conserved Surface Amino Acids in Binding of SmpB Protein to SsrA RNA

Bacteria possess a unique salvage mechanism for rescuing ribosomes stalled on aberrant mRNAs. A complex of SmpB protein and SsrA RNA orchestrates this salvage process. The specific and direct binding of SmpB facilitates recognition and delivery of SsrA RNA to stalled ribosomes. The SmpB protein is conserved throughout the bacterial kingdom and contains several conserved amino acid sequence motifs. We present evidence to demonstrate that amino acid residues Glu-31, Leu-91, and Lys-124, which are highly conserved and clustered along an exposed surface of the protein, play a crucial role in the SsrA-mediated peptide tagging process. Our analysis suggests that the peptide-tagging defect exhibited by these SmpB variants is due to their inability to facilitate the delivery of SsrA RNA to stalled ribosomes. Moreover, we present evidence to demonstrate that the ribosome association defect of these variants is due to their reduced SsrA binding affinity. Consistent with these findings, we present biochemical evidence to demonstrate that residues Glu-31, Leu-91, and Lys-124 are essential for the SsrA binding activity of SmpB protein. Furthermore, we have investigated the interactions of SmpB.SsrA orthologues from the thermophilic bacterium Thermoanaerobacter tengcongensis. Our investigations demonstrate an analogous role for the equivalent T. tengcongensis residues in SmpB.SsrA interactions, hence further validating our findings for the Escherichia coli SmpB.SsrA system. These results demonstrate the functional significance of this cluster of conserved residues in SmpB binding to SsrA RNA, suggesting they might represent a core contact surface for recognition of SsrA RNA.

Mechanisms of Helicases

Mechanisms of Helicases

A Blight Upon Malaria

CELL BIOLOGY Many pathogens, whether of plants or animals, export their own proteins in order to modify their local environment and to disable host defenses. Channeling secretory proteins into the molecular machines that allow them to cross membranes generally relies on short amino acid sequence motifs. Oomycetes are deep-branching eukaryotes that include the notorious Phytophthora , which causes devastating diseases such as potato late blight and sudden oak death. Phytophthora colonizes plants by establishing and residing within a membrane-bounded haustorium inside cells. Bhattacharjee et al. show that Plasmodium and Phytophthora , phylogenetically distant eukaryotic pathogens, use similar host-targeting motifs. Plasmodium has a host-targeting motif that is linked to over 400 virulence factors, which make up the “secretome.” Phytophthora possesses virulence determinants that are recognized by plant hosts and, like Plasmodium, the highly conserved leader sequence motif RxLR along with a nearby acidic domain can be found in multiple effectors that are delivered into the host cell cytoplasm. — CA PloS Pathol. 2 , e50 (2006).

The transthyretin-related protein: Structural investigation of a novel protein family

The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 Å resolution in the presence of zinc, and the other at 2.1 Å resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.

Amino acid sequence motifs and mechanistic features of the membrane translocation of α‐synuclein

Many lines of evidence suggest that alpha-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of alpha-synuclein, and the mechanistic features of the phenomenon. We first showed that not only alpha-synuclein but also beta- and gamma-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of alpha-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. alpha-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of alpha-synuclein at 4 degrees C was comparable with the import observed at 37 degrees C. Furthermore, membrane translocation of alpha-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of alpha-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.

EXPRESSION OF A FUNCTIONAL CANINE CYTOCHROME b5 REDUCTASE

Genomic sequences derived from diverse eukaryotic species have greatly increased the number of putative cytochrome b5 reductase (cb5r) sequences. BLAST searches have revealed several Canis familiaris cb5r or DIA1 gene products. However, alignments with known cb5r sequences indicated structural anomalies, including N-terminal and internal sequence variations. To confirm the canine sequence, we utilized PCR primers derived from a mammalian consensus sequence to clone and express the functional protein from beagle lung tissue. The canine cb5r cDNA sequence comprised 906 bp coding for 300 amino acid residues with 93% and 87% sequence identity to both human and rat cb5r’s, respectively, and retained all the characteristic amino acid sequence motifs associated with members of the flavoprotein transhydrogenase superfamily. Expression of the soluble canine cb5r diaphorase domain (I33-F300) resulted in a FAD-containing protein of mass (m/z) 31,505 (31,522 calc.) with spectroscopic properties identical to other mammalian cb5r’s, NADH:ferricyanide reductase activity (kcat=767 s−1, Km=7 μM NADH), a marked specificity for NADH vs. NADPH, a redox potential (n=2) for the FAD prosthetic group of −274 mV and binding constants for H4NAD (67 μM) and NAD+ (787 μM). These results identify the first correct sequence for C. familiaris cb5r and indicate a protein structurally and functionally comparable to other mammalian variants (supported by NIH).

Identification of the Flavin Binding Site in the Flavin Reductase from the Alkanesulfonate Monooxygenase System in Escherichia coli

Sulfate starvation in Escherichia coli increases the synthesis of specific proteins which utilize alternate sulfur sources for growth. A tow-component enzyme system has been show to be involved in the conversion of alkanesulfonates to sulfides and the corresponding aldehyde. The system consists of two proteins, a flavin reductase (SsuE) and a monooxygenase (SsuD) which together catalyze the oxidation of alkanesulfonate to the products. Previous studies have identified a conserved amino acid sequence, RXFS, from an alignment search of E. coli flavin reductases. The Ser residue has been shown to be involved in flavin binding by interacting with the isoalloxazine ring at N(5). An alignment search of SsuE also identified a similar conserved amino acid sequence motif, R51FDS54, which may be involved in flavin binding. Ser54 from wild type SsuE was substituted with alanine and the SsuES54A protein purified. Results from circular dichroism spectroscopy of SsuES54A were essentially identical to wild type SsuE suggesing there were no major secondary structural changes due to this mutation. The km and kcat values for SsuES54A were 0.09 μM and 101.83 min−1 respectively. Compared with previous results from wild type SsuE, the km increased by 6-fold and the catalytic efficiency kcat/km decreased by 6.4-fold. In addition, the different kd value for SsuES54A compared to the wild type protein suggested this mutation altered the affinity of flavin binding. Our results show that Ser54 plays an essential role in flavin binding to SsuE. To further probe the flavin binding sites, studies involving multiple mutations of the conserved R51FDS54 region will be performed. This research is supported by Auburn University.

Functions of Hel308 helicases in promoting genome stability in metazoans and archaea

Hel308 helicase promotes genome stability in Drosophila and mouse, and in the third domain of life, Archaea. This enzyme is conserved throughout metazoans and archaea but is absent from bacteria and yeasts. Very little is known about how Hel308 functions, but we have characterised the action of archaeal Hel308 (Hel308a), showing that it targets lagging strands for dissociation from model replication forks. We also show that Hel308a also interacts with replication forks in vivo. We describe how Hel308a has low apparent processivity and favours unwinding of lagging strands from a fork branchpoint with a ssDNA gap. Unwinding by Hel308a is reminiscent of bacterial PriA and Rep helicases. We describe an amino acid sequence motif invariant at the C-terminus of all Hel308 proteins that is distinct from typical helicase motifs, and is required for ATP hydrolysis. Preliminary data will be presented, suggesting that Hel308a antagonises RadA, the archaeal Rad51 protein. Yeast two-hybrid analysis of hel308 from archaea against genome libraries has identified interacting partners that may shed light on the DNA repair/replication context in which Hel308 functions. This research was supported by a fellowship to ELB from The Wellcome Trust.

Mutational Analysis of the Catalytic Domain of the Murine Dnmt3a DNA-(cytosine C5)-methyltransferase

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.

Hin4II, a new prototype restriction endonuclease from Haemophilus influenzae RFL4: Discovery, cloning and expression in Escherichia coli

The genes encoding restriction-modification system of unknown specificity Hin4II from Haemophilus influenzae RFL4 were cloned in Escherichia coli and sequenced. The Hin4II system comprises three tandemly arranged genes coding for m6A DNA methyltransferase, m5C DNA methyltransferase and restriction endonuclease, respectively. Restriction endonuclease was expressed in E. coli and purified to apparent homogeneity. The DNA recognition sequence and cleavage positions were determined. R.Hin4II recognizes the novel non-palindromic sequence 5'-CCTTC-3' and cleaves the DNA 6 and 5 nt downstream in the top and bottom strand, respectively. The new prototype restriction endonuclease Hin4II was classified as a potential candidate of HNH nuclease family after comparison against SMART database. An amino acid sequence motif 297H-X14-N-X8-H of Hin4II was proposed as forming a putative catalytic center.

Molecular Determinants of Kinase Pathway Activation by Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) mainly activates programmed cell death through caspases. By contrast, TNF primarily induces gene transcription through the inhibitor of kappaB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. Apo2L/TRAIL also can stimulate these kinases, albeit less strongly; however, the underlying mechanisms of this stimulation and its relation to apoptosis are not well understood. Here we show that Apo2L/TRAIL activates kinase pathways by promoting the association of a secondary signaling complex, subsequent to assembly of a primary, death-inducing signaling complex (DISC). The secondary complex retained the DISC components FADD and caspase-8, but recruited several factors involved in kinase activation by TNF, namely, RIP1, TRAF2, and NEMO/IKKgamma. Secondary complex formation required Fas-associated death domain (FADD), as well as caspase-8 activity. Apo2L/TRAIL stimulation of JNK and p38 further depended on RIP1 and TRAF2, whereas IKK activation required NEMO. Apo2L/TRAIL induced secretion of interleukin-8 and monocyte chemoattractant protein-1, augmenting macrophage migration. Thus, Apo2L/TRAIL and TNF organize common molecular determinants in distinct signaling complexes to stimulate similar kinase pathways. One function of kinase stimulation by Apo2L/TRAIL may be to promote phagocytic engulfment of apoptotic cells.

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

The bacterial flagellum is a predominantly cell-external super-macromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhB(C)), which is consistently and specifically cleaved into two subdomains (FlhB(CN) and FlhB(CC)) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhB(C)(P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB(C) and FlhB(C)(P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.

Small Ubiquitin-like Modifier (SUMO) Recognition of a SUMO Binding Motif: A REVERSAL OF THE BOUND ORIENTATION

Sumoylation has recently been identified as an important mechanism that regulates protein interactions and localization in essential cellular functions, such as gene transcription, subnuclear structure formation, viral infection, and cell cycle progression. A SUMO binding amino acid sequence motif (SBM), which recognizes the SUMO moiety of modified proteins in sumoylation-dependent cellular functions, has been consistently identified by several recent studies. To understand the mechanism of SUMO recognition by the SBM, we have solved the solution structure of SUMO-1 in complex with a peptide containing the SBM derived from the protein PIASX (KVDVIDLTIESSSDEEEDPPAKR). Surprisingly, the structure reveals that the bound orientation of the SBM can reverse depending on the sequence context. The structure also reveals a novel mechanism of recognizing target sequences by a ubiquitin-like module. Unlike ubiquitin binding motifs, which all form helices and bind to the main beta-sheet of ubiquitin, the SBM forms an extended structure that binds between the alpha-helix and a beta-strand of SUMO-1. This study provides a clear mechanism of the SBM sequence variations and its recognition of the SUMO moiety in sumoylated proteins.

Restricted Pairing of Immunoglobulin Heavy and Light Chains Expressed by Chronic Lymphocytic Leukemia B Cells Is Predicated on the Heavy Chain CDR3.

Analysis of the immunoglobulin (Ig) heavy chains expressed by the leukemic B cells of patients with chronic lymphocytic leukemia (CLL) has demonstrated that expression of Ig variable heavy chain (VH) genes in CLL is not random. Certain VH genes are more frequently expressed in CLL than in the normal adult B cell repertoire, and some, such as the 51p1 allele of VH1-69, also use of certain diversity (D) and junctional (JH) gene segments that encode third complementarity determining regions (CDR3) with conserved molecular structures. We identified 15 CLL cases among 1,220 examined that express nearly identical Ig heavy and light chains, encoded by 51p1/D3-16/JH3 and VKA27, respectively (Blood , 104:2499, 2004). The highly restricted and virtually identical structure of these B cell receptors strongly suggests selection for Ig in CLL that have a particular binding activity. However, little information is currently available about the light chains expressed by CLL B cells that have 51p1-encoded Ig heavy chains that use other D and JH segments encoding CDR3 that also are repeatedly observed in this disease. We analyzed the VL genes used by 235 CLL cases found to express 51p1-encoded Ig heavy chains among 1,605 CLL patients examined. First, we find restricted light chain isotype expression, as 72% of samples express kappa and 28% express lambda light chains, compared to 65% kappa and 35% lambda within the cohort of all 1,605 CRC CLL samples, and about 60% kappa and 40% lambda expression in normal blood B cells. Nucleotide sequence analysis of the Ig light chain V gene used by these 235 cases revealed that each had greater than 98% homology to an identified germline VK or Vl gene. Additionally, we identified non-stochastic pairing of particular VK and Vl genes with 51p1-encoded heavy chains that have highly-conserved CDR3. Twenty of the 235 cases (8.5%) were found to have Ig light chains encoded by VKO2. Seventeen (85%) of such cases had 51p1-encoded Ig heavy chains that used D2-2 and JH6, 15 of which had nearly identical CDR3 using the amino acid motif DIVVVPAAI. The VKO2-encoded light chains paired with these Ig heavy chains all had nearly identical CDR3 with the amino acid sequence motif QQSYSTPRT. Similarly, seven of the 235 cases (3%) were found to have Ig light chains encoded by Vl3-9. Six (86%) of such cases had 51p1-encoded Ig heavy chains that used D3-3 and JH6, and all had highly conserved heavy chain CDR3 with the amino acid motif YDFWSGYYPNYYYYGMDV. The Vl3-9-encoded light chains paired with these Ig heavy chains all had nearly identical CDR3 with the amino acid sequence motif QVWDSSTXV. Finally, we identified seven additional samples that express a heavy chain using D3-16 and JH3 that have nearly identical CDR3 amino acid sequences GGGYDYIWGSYRPNDAFDI, and also express light chains encoded by VKA27. These seven samples combined with the previous 15 represent all of the 51p1-encoded heavy chains that utilize D3-16 and JH3, as well as 52% (22 of 42) of all 51p1-encoded CLL samples that express VKA27-encoded light chains. These studies reveal for the first time that CLL cases using the same unmutated Ig heavy chain have non-stochastic pairing with disparate Ig light chains that is predicated upon the Ig heavy chain CDR3 structure. Because the CDR3 typically forms a major part of antibody binding site(s) for antigen, these data provide compelling evidence for antigen selection of the antibodies expressed in CLL.

Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449

The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 4'-O-carbamoyl-2'-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA- from methoxymalonyl-ACP-specific AT domains in natural PKSs.