Polypeptide Linker Research Articles

Single Point Mutation in Bin/Amphiphysin/Rvs (BAR) Sequence of Endophilin Impairs Dimerization, Membrane Shaping, and Src Homology 3 Domain-mediated Partnership

Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are essential players in the dynamics of intracellular compartments. The BAR domain is an evolutionarily conserved dimeric module characterized by a crescent-shaped structure whose intrinsic curvature, flexibility, and ability to assemble into highly ordered oligomers contribute to inducing the curvature of target membranes. Endophilins, diverging into A and B subgroups, are BAR and SH3 domain-containing proteins. They exert activities in membrane dynamic processes such as endocytosis, autophagy, mitochondrial dynamics, and permeabilization during apoptosis. Here, we report on the involvement of the third α-helix of the endophilin A BAR sequence in dimerization and identify leucine 215 as a key residue within a network of hydrophobic interactions stabilizing the entire BAR dimer interface. With the combination of N-terminal truncation retaining the high dimerization capacity of the third α-helices of endophilin A and leucine 215 substitution by aspartate (L215D), we demonstrate the essential role of BAR sequence-mediated dimerization on SH3 domain partnership. In comparison with wild type, full-length endophilin A2 heterodimers with one protomer bearing the L215D substitution exhibit very significant changes in membrane binding and shaping activities as well as a dramatic decrease of SH3 domain partnership. This suggests that subtle changes in the conformation and/or rigidity of the BAR domain impact both the control of membrane curvature and downstream binding to effectors. Finally, we show that expression, in mammalian cells, of endophilin A2 bearing the L215D substitution impairs the endocytic recycling of transferrin receptors.

Metal ion and DNA binding by single-chain PvuII endonuclease: lessons from the linker

Understanding the roles of metal ions in restriction enzymes has been complicated by both the presence of two metal ions in many active sites and their homodimeric structure. Using a single-chain form of the wild-type restriction enzyme PvuII (scWT) in which subunits are fused with a short polypeptide linker (Simoncsits et al. in J. Mol. Biol. 309:89-97, 2001), we have characterized metal ion and DNA binding behavior in one subunit and examined the effects of the linker on dimer behavior. scWT exhibits heteronuclear single quantum coherence NMR spectra similar to those of native wild-type PvuII (WT). For scWT, isothermal titration calorimetry data fit to two Ca(II) sites per subunit with low-millimolar K (d)s. The variant scWT|E68A, in which metal ion binding in one subunit is abolished by mutation, also binds two Ca(II) ions in the WT subunit with low-millimolar K (d)s. When there are no added metal ions, DNA binding affinity for scWT is tenfold stronger than that of the native WT, but tenfold weaker at saturating Ca(II) concentration. In the presence of Ca(II), scWT|E68A binds target DNA similarly to scWT, indicating that high-affinity substrate binding can be carried energetically by one metal-ion-binding subunit. Global analysis of DNA binding data for scWT|E68A suggests that the metal-ion-dependent behaviors observed for WT are reflective of independent subunit behavior. This characterization provides an understanding of subunit contributions in a homodimeric context.

Bifunctional fusion proteins of the human engineered antibody domain m36 with human soluble CD4 are potent inhibitors of diverse HIV-1 isolates

Currently used antiretroviral therapy is highly successful but there is still a need for new effective and safe prophylactics and therapeutics. We have previously identified and characterized a human engineered antibody domain (eAd), m36, which exhibits potent broadly neutralizing activity against HIV-1 by targeting a highly conserved CD4 binding-induced (CD4i) structure on the viral envelope glycoprotein (Env) gp120. m36 has very small size (∼15 kDa) but is highly specific and is likely to be safe in long-term use thus representing a novel class of potentially promising HIV-1 inhibitors. Major problems with the development of m36 as a candidate therapeutic are possible short serum half life and lack of effector functions that could be important for effective protection in vivo. Fusion of m36 to human IgG1 Fc resulted in dramatically diminished neutralization potency most likely due to the sterically restricted nature of the m36 epitope that limits access of large molecules. To confer effector functions and simultaneously increase the potency, we first matured m36 by panning and screening a mutant library for mutants with increased binding to gp120. We next fused m36 and its mutants with the first two domains (soluble CD4, sCD4) of the human CD4 using a polypeptide linker. Our results showed that the selected m36 mutants and the sCD4 fusion proteins exhibited more potent antiviral activities than m36. The m36-sCD4 fusion proteins with human IgG1 Fc showed even higher potency likely due to their bivalency and increased avidity although with a greater increase in molecular size. Our data suggest that m36 derivatives are promising HIV-1 candidate therapeutics and tools to study highly conserved gp120 structures with implications for understanding mechanisms of entry and design of vaccine immunogens and small-molecule inhibitors.

Highly Enhanced Cytotoxicity of a Dimeric Bispecific Diabody, the hEx3 Tetrabody

We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.

SCD4-17b bifunctional protein: extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates.

BackgroundWe previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types.ResultsWe observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses.ConclusionsThe results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.

Efficient production of single-chain Fv antibody possessing rare codon linkers in fed-batch fermentation

Single-chain Fv antibody (scFv) having 2 types of polypeptide linkers with or without rare codons, namely scFv (G(4)S)(3)(R) and scFv No.10 (with rare codons) and scFv (G(4)S)(3) and scFv No.10(NR) (without rare codon), were expressed under controllable conditions in batch and fed-batch fermentation, in order to compare volumetric productivity and specific productivity levels of scFvs as a soluble form. In batch fermentation, volumetric productivity levels of scFv (G(4)S)(3)(R) and scFv No.10, namely scFvs having the rare codon linkers were 3-5 times higher than those of scFvs that had linkers without the rare codon. In fed-batch fermentation controlled by an exponential feeding system, the cell concentrations of the transformants increased with similar specific growth rates (0.1 h(-1)), while the specific productivity levels of scFvs with the rare codon linkers were 1.6 times higher than those of scFvs without the rare codon linkers. These results indicate that the presence of several rare codons in the gene of a polypeptide linker increases soluble amount of scFvs. This might be caused by a temporary decrease in translation speed at the position of the polypeptide linker allowing time for the folding of the V(H) domain and avoiding unfavorable interactions between amino acid residues at the unfolded V(H) and V(L) domains. Higher specific productivity levels of both scFv No. 10 and scFv No. 10(NR) than those of scFv (G(4)S)(3)(R) and (G(4)S)(3) might be caused by difference in stability of the polypeptide linkers on the basis of amino acid sequences. Thus, the rare codon linkers tested in this study will be considerably useful for large-scale production of soluble and active scFvs in fed-batch or continuous fermentations, in which high cell activity can be maintained.

Monitoring Caspase-3 Activation with a Multimodality Imaging Sensor in Living Subjects

Capsase-3 plays an important role in chemotherapy-induced apoptosis in many cancers. Herein, we applied a multimodality reporter vector to monitor caspase-3 activation indirectly in live cells and tumors of living animals undergoing apoptosis. A fusion protein (MTF) was constructed by combining three different reporter proteins, red fluorescent protein (mRFP1), firefly luciferase (FL), and HSV1-sr39 truncated thymidine kinase (TK), linked through a caspase-3 recognizable polypeptide linker. After cleavage by caspase-3, a significant gain in mRFP1, FL, and TK activity are observed by fluorescence-activated cell sorting and enzyme-based assays. A melanoma cell line (B16F10-mtf-hrl) stably expressing mtf (to measure caspase-3 activation) and hrl-IRES-gfp (to determine the decrease in a number of viable cells) vectors was generated to measure two independent molecular events upon treatment. Upon induction with 8 mumol/L staurosporine, the fusion protein showed a 2.8-fold increase in FL (P = 0.03), a 1.5-fold increase in TK (P = not significant), and a 2-fold increase in mRFP1 (P = 0.05) activity in 293T cells. Bioluminescence and micropositron emission tomography imaging of the apoptotic B16F10-mtf-hrl tumors showed a 2-fold higher FL activity (897 versus 416) and a 2-fold higher TK activity (10.3 versus 3.87) than control tumors when normalized with RL activity. Using a similar normalization approach, the time kinetics of caspase-3 activation by two protein kinase-C inhibitors was noninvasively monitored in living mice. This multimodality caspase sensor vector could effectively and noninvasively monitor caspase-3 activation from single live cells to a multicellular tumor environment and, thus, would be a valuable tool for drug screening in preclinical models and future patient cell based therapy.

Small Angle X-ray Studies Reveal That Aspergillus niger Glucoamylase Has a Defined Extended Conformation and Can Form Dimers in Solution

The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.

Effect of Nucleotide Sequences of Polypeptide Linkers on Production of Soluble Single-Chain Fv Antibodies

Effects of nucleotide sequences of polypeptide linkers on production of soluble single-chain Fv antibodies in E. coli were studied using scFv genes, in which genes of VH and VL domains were genetically linked with 4 types of linker genes with or without rare codons, namely genes of scFv (G4S)3, scFv (G4S)3R, scFv No. 10 and scFv No. 10NR. The transformants expressing scFvs having linkers with 3–4 rare codons (scFv (G4S)3R and scFv No. 10) could grow to higher cell concentrations under induction condition, while growth of the cells expressing scFvs having linkers without rare codon (scFv (G4S)3 and scFv No. 10NR) stopped after 3 h. Productivity levels of soluble scFv by cells expressing scFv (G4S)3R and scFv No. 10 were higher than those of scFv (G4S)3 and scFv No. 10NR. The specific productivity levels of inclusion bodies for scFv (G4S)3R and scFv No. 10 decreased in comparison with those for scFv (G4S)3 and scFv No. 10NR. This might show that decrease in the translation rate at the region coding polypeptide linkers with rare codons has a favorable effect for the subsequent folding process of scFv molecules. Furthermore, from a SPR analysis using an antigen-immobilized sensor chip, scFv (G4S)3R showed the same binding affinity as the original scFv (G4S)3. Thus, nucleotide sequences of polypeptide linkers are important to increase productivity of scFv expressed in soluble fraction. The nucleotide sequence of the flexible linker (G4S)3R used in this study may be useful for efficient production of various scFvs without loss of the binding affinity of target scFv.

Extended Polypeptide Linkers Establish the Spatial Architecture of a Pyruvate Dehydrogenase Multienzyme Complex

Icosahedral pyruvate dehydrogenase (PDH) enzyme complexes are molecular machines consisting of a central E2 core decorated by a shell of peripheral enzymes (E1 and E3) found localized at a distance of approximately 75-90 A from the core. Using a combination of biochemical, biophysical, and cryo-electron microscopic techniques, we show here that the gap between the E2 core and the shell of peripheral enzymes is maintained by the flexible but extended conformation adopted by 60 linker polypeptides that radiate outwards from the inner E2 core, irrespective of the E1 or E3 occupancy. The constancy of the gap is thus not due to protein-protein interactions in the outer protein shell. The extended nature of the E2 inner-linker regions thereby creates the restricted annular space in which the lipoyl domains of E2 that carry catalytic intermediates shuttle between E1, E2, and E3 active sites, while their conformational flexibility facilitates productive encounters.

Phage display of chelating recombinant antibody libraries

High affinity and discriminating specificity are important parameters for any successful antibody based targeting strategy. We herein describe a system for the construction and subsequent selection of affinity-optimised chelating recombinant antibodies (CRAbs) from a randomised filamentous phage-display inter-scFv linker library. Using a simple, robust and highly degenerate tandem scFv cloning strategy a phage-display library of CRAbs with varied inter-scFv linkers was constructed and characterised. The library consisted of two single-chain Fvs (scFvs) of well characterised anti-lysozyme antibodies D1.3 and HyHEL-10 TF, specific for distinct non-overlapping epitopes, separated by flexible polypeptide linkers of varying lengths and sequences. The use of a stringent affinity-based selection strategy quickly led to the enrichment of CRAbs with a restricted set of linker lengths (16–21 amino acids) which agrees very closely with previously described crystal structure data, affinity measurements and mathematical modelling. This CRAb linker phage-display selection strategy is a widely applicable approach for the selection of very high affinity CRAbs for pairs of scFvs against potentially any target antigen, complementing the more arbitrary affinity maturation approaches based on random mutagenesis.

Single chain Fab (scFab) fragment.

BackgroundThe connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.ResultsHere, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised.ConclusionA new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.

Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase.

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C(pro) and the viral polymerase 3D(pol) and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-A resolution and the G64S fidelity mutant of 3D(pol) at a 3.0-A resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D(pol) G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D(pol) makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

Polypeptide linkers suitable for the efficient production of dimeric scFv in Escherichia coli

Single chain variable fragment antibodies (scFvs) were selected from a phage library displaying scFvs consisting of the VH and VL domains of an anti-bisphenol A monoclonal antibody and 20 randomized amino acids linkers by biopanning using an antigen bisphenol A. After four rounds of biopanning selections, seven scFvs with different polypeptide linkers were isolated. The scFvs expressed by the Escherichia coli transformants predominantly formed dimeric structures, and were found in soluble fractions at higher concentrations than those of scFvs containing flexible linkers (G 4S) 1–3, because the transformants expressing these scFvs could grow to higher cell concentration after the induction of scFv production with IPTG. Consequently, five times higher productivities of soluble scFvs were attained compared with scFvs having the flexible linkers. The antigen binding activities of these scFvs were confirmed by affinity chromatography using an antigen-coupled column and ELISA. The selected polypeptide linkers will be useful for the production of soluble scFv dimers in E. coli.

A central role for extended polypeptide linkers in establishing the spatial architecture of a pyruvate dehydrogenase complex

The pyruvate dehydrogenase complex is among the largest multifunctional enzymes found in cells (>11MDa). It catalyzes the production of acetyl-CoA from pyruvate and is comprised of three component enzymes: E1, E2, and E3. Previous work from our laboratory resulted in determination of the molecular architectures of sub-complexes comprising the 60-mer E2 from Bacillus stearothermophilus decorated with either 60 copies of E1 or E3. An annular gap of ~75–95 Å separates the catalytic domains of the E2 from an outer shell formed of E1’s or E3’s. To test the mechanisms involved in the maintenance of this annular gap and its significance for active site coupling, cryo-electron tomography was employed to determine the spatial relationship of E1 and E2 components within individual, non-averaged, E1E2 sub-complexes. In addition to verifying the presence of the annular space in three dimensions, stochastic variation in the exact position of each individual E1 density relative to the E2 core was measured. Biophysical studies of a peptide, corresponding to the inner linker region, suggests this region is flexible, yet highly extended, and maintains a long persistence length (~70–89 Å). Together these results demonstrate that the inner linker region separating the bound E1 and E3 molecules from the E2 core is an extended peptide chain that maintains a long persistence length, sixty of which generate a scaffold for the organization of E1 and E3 molecules in a modular radial shell. This is likely to be critically important to the coupling of E1, E2, and E3 active sites, mediated by movements across the gap by an additional domain at the N-terminal end of the E2 chain.

Conformation of Polypyrimidine Tract Binding Protein in Solution

The polypyrimidine tract binding protein (PTB) is an RNA binding protein that normally functions as a regulator of alternative splicing but can also be recruited to stimulate translation initiation by certain picornaviruses. High-resolution structures of the four RNA recognition motifs (RRMs) that make up PTB have previously been determined by NMR. Here, we have used small-angle X-ray scattering to determine the low-resolution structure of the entire protein. Scattering patterns from full-length PTB and deletion mutants containing all possible sequential combinations of the RRMs were collected. All constructs were found to be monomeric in solution. Ab initio analysis and rigid-body modeling utilizing the high-resolution models of the RRMs yielded a consistent low-resolution model of the spatial organization of domains in PTB. Domains 3 and 4 were found to be in close contact, whereas domains 2 and especially 1 had loose contacts with the rest of the protein.

Modulation of Single-Chain Antibody Affinity with Temperature-Responsive Elastin-Like Polypeptide Linkers

Single-chain antibodies are genetically engineered constructs composed of a VH and VL domain of an antibody linked by a flexible peptide linker, commonly (GGGGS)3. We asked whether replacement of this flexible linker with peptides known to undergo environmentally induced structural transitions could lead to antibodies with controlled binding and release characteristics. To this end, we genetically modified and produced a series of anti-fluorescein single-chain antibodies with the general linker sequence (VPGXG)n, where n is 1.2 to 3 and X is Val or His, to evaluate the effects of linker length and composition. Our results indicate that single-chain antibodies containing elastin-like polypeptide linkers have equilibrium affinity (KD) comparable to wild-type (GGGGS)3 at room temperature but altered binding kinetics and faster ligand release as the temperature is raised. These results are consistent with the increased molecular order and contraction that elastin-like polypeptides are known to undergo with increased temperature. Modulation of antibody affinity using stimulus-responsive linkers may have applications in biosensors, drug delivery, and bioseparations.

High efficient mammalian expression and secretion of a functional humanized single-chain Fv/human interleukin-2 molecules

To construct and produce a recombinant bispecific humanized single-chain Fv (sFv) /Interleukin-2 (IL-2) fusion protein by using mammalian cells. The sFv/IL-2 protein was genetically engineered, and transfected to mammalian cells to determine whether the mammalian protein folding machinery can produce and secrete active sFv/IL-2 with high efficiency. The fusion protein was constructed and high efficiently expressed with yields up to 102 +/- 4.2 mg/L in culture supernatant of the stably transfected 293 cell line. This recombinant fusion protein consisted of humanized variable heavy (V(H)) and light (V(L)) domains of monoclonal antibody (mAb) 520C9 directed against the human HER-2/neu (c-erbB2) proto-oncogene product p185, and human IL-2 connected by polypeptide linker. The fusion protein was shown to retain the immunostimulatory activities of IL-2 as measured by IL-2-dependent cell proliferation and cytotoxicity assays. In addition to its IL-2 activities, this fusion protein also possessed antigen-binding specificity against p185, as determined by indirect ELISA using p185 positive SKOV 3ip1 cells. The large-scale preparation of the recombinant humanized sFv antibody/IL-2 fusion protein is performed with 293 cells. The recombinant humanized sFv antibody/IL-2 fusion protein may provide an effective means of targeting therapeutic doses of IL-2 to p185 positive tumors without increasing systemic toxicity or immunogenicity.

Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

Solution-state NMR investigation of DNA binding interactions in Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg): a dynamic description of the DNA/protein interface

Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair (BER) protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free in solution and bound to a double-stranded DNA oligomer containing 1,3-propanediol (13-PD), a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 251 (83%) free-precession 15N/ 1H HSQC cross peaks were observed and 180 of these were assignable, indicating that ∼30% of the residues undergo intermediate motion on the NMR timescale, broadening the resonances beyond detection or making them intractable in backbone assignment experiments. The majority of these affected residues were in the polypeptide linker region and the interface between the N- and C-terminal domains. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate timescale motion observed for free Fpg, including those residues directly involved in DNA binding, notwithstanding a nanomolar dissociation constant for 13-PD binding. Indeed, after binding to 13-PD, at least ∼40% of the Fpg residues undergo intermediate timescale motion even though all other residues exhibit tight DNA binding characteristic of slow exchange. CPMG–HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that were quenched upon binding 13-PD. In free Fpg, heteronuclear 1H– 15N NOE experiments detected picosecond timescale backbone motion in the αF–β9 loop, the region primarily responsible for chemically discriminating 8-oxoguanine (8-oxoG) over normal guanine, that was quenched after binding 13-PD. Collectively, these observations reveal that, in solution, Fpg is a very dynamic molecule even after binding damaged DNA. Such motion, especially at the DNA binding surface, may be key to its processive search for DNA damage and its catalytic functions once it recognizes damaged DNA.