Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond

Almog, Orna; Benhar, Itai; Vasmatzis, George; Tordova, Maria; Lee, Byung Kook; Pastan, Ira; Gilliland, Gary L.

doi:10.1002/(sici)1097-0134(19980501)31:2<128::aid-prot3>3.0.co;2-i

Cited by 17 publications

(5 citation statements)

References 35 publications

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“…Our results are in contrast to most structural studies with engineered disulfide bonds where normally significant deviations around the disulfide linkage are found (Jacobson et al 1992; Wakarchuk et al 1994; Almog et al 1998). The domain rotation of 5′‐NT might assist to accommodate the local packing restraints imposed by the inserted cross‐link by augmenting the flexibility of the disulfide bond.…”

Section: Resultscontrasting

confidence: 99%

Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges

2004

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Engineering disulfide bridges is a common technique to lock a protein movement in a defined conformational state. We have designed two double mutants of Escherichia coli 5Ј-nucleotidase to trap the enzyme in both an open (S228C, P513C) and a closed (P90C, L424C) conformation by the formation of disulfide bridges. The mutant proteins have been expressed, purified, and crystallized, to structurally characterize the designed variants. The S228C, P513C is a double mutant crystallized in two different crystal forms with three independent conformers, which differ from each other by a rotation of up to 12°of the C-terminal domain with respect to the N-terminal domain. This finding, as well as an analysis of the domain motion in the crystal, indicates that the enzyme still exhibits considerable residual domain flexibility. In the double mutant that was designed to trap the enzyme in the closed conformation, the structure analysis reveals an unexpected intermediate conformation along the 96°rotation trajectory between the open and closed enzyme forms. A comparison of the five independent conformers analyzed in this study shows that the domain movement of the variant enzymes is characterized by a sliding movement of the residues of the domain interface along the interface, which is in contrast to a classical closure motion where the residues of the domain interface move perpendicular to the interface.Keywords: X-ray crystallography; 5Ј-nucleotidase; protein engineering; disulfide trapping; disulfide geometry; domain rotation; protein movement; protein flexibility; TLS refinement; protein conformation Many extracellular proteins contain disulfide bridges that provide the protein with extra rigidity (Thornton 1981). Consequently, the stability of proteins can be greatly enhanced with the help of genetically introduced disulfide bridges, including an improved thermal stability (Velanker et al. 1999;Almog et al. 2002). Engineering disulfide bonds into proteins became an important tool for an analysis of the role of protein motions for enzyme catalysis (Matsumura and Matthews 1989), and in general, for detecting functional protein conformational changes (Lee et al. 1995;Kawate and Gouaux 2003). Trapping of a protein motion does not only demonstrate the importance of the movement but also enables an analysis of individual protein conformers in solution where otherwise different conformational states are likely to be present. The presence of the open and closed forms of the enzyme in equilibrium has been demonstrated for citrate synthase, for which both an open and a closed conformer were crystallographically observed, even in crystals grown in the same crystallization drop (Liao et al. 1991). Locking a protein in a specific conformation may allow for an unambiguous assignment of experimental signals (e.g., from spectroscopic studies) to the respective conformation.In Escherichia coli 5Ј-nucleotidase (5Ј-NT), a versatile enzyme that hydrolyses mono-, di-, and trinucleotides as Reprint requests to: Norbert Sträter, Biotec...

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Section: Resultscontrasting

confidence: 99%

Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges

2004

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“…In a recent study, van Roon et al described structures for the 291-2G3-A Fab in the unliganded state and in complex with the Le x β-O-methyl glycoside at 2.05 and 1.8 Å resolution, respectively. 49 Crystal structures for antigen binding fragments have been determined for two additional antibodies specific for Le y (B1) 50 and Le a (CF4C4), 51 but both of these are in the unliganded state and will not be discussed further.…”

Section: Antibody Complexes With Lewis Carbohydrate Ligandsmentioning

confidence: 99%

Three‐dimensional structures of carbohydrate determinants of Lewis system antigens: Implications for effective antibody targeting of cancer

et al. 2005

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Summary Lewis system carbohydrate antigens have been shown to be expressed at high levels in many cancers of epithelial cell origin, including those of colon, breast, lung, prostate and ovary. The type 1 (Le a and Le b ) antigens are important histo-blood groups, while type 2 (Le x and Le y ) antigens in healthy individuals are only expressed, at relatively low levels, by a few tissues, including some epithelial cells. Thus, the type 2 antigens are considered to be tumour-associated antigens and are promising targets for cancer treatment, including antibody-based immunotherapy. In this review, we discuss the conformational characteristics of the free and bound forms of Lewis oligosaccharides and the 3D structures of antibodies in complex with Le y and Le x antigens. Collectively, the structural studies have demonstrated that the Lewis determinants are rigid structures, which generally maintain the same conformation in the free and bound states. The rigid nature and similarities in shape of type 1 and 2 Lewis oligosaccharides appear to make them perfectly suited to driving a structurally convergent immune response (at least in the case of Le y specific antibodies) toward a highly specific recognition of individual carbohydrate determinants, which is a goal in the development of effective antibody-based cancer treatments.

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“…It usually forms by the coupling of two mercapto groups 3 . In biology, the disulfide bond is an important part of the secondary and tertiary structures of proteins, which is formed between the mercapto groups of cysteine residues 4,5 . It plays an important role in the formation of the three‐dimensional structure of protein molecules 6,7 .…”

Section: Introductionmentioning

confidence: 99%

Fabrication and characterization of glutathione‐responsive nanoparticles from the disulfide bond‐bridged block copolymer

Shi

Yang

Pan

et al. 2021

Polymers for Advanced Techs

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The purpose of this paper is to fabricate novel nanoparticles (NPs) from a single disulfide bond‐bridged block copolymer poly(hydroxyethyl methacrylate)‐S‐S‐polycaprolactone (PHEMA‐S‐S‐PCL). The novel biomaterial was synthesized by ring‐opening polymerization and reversible addition–fragmentation chain transfer polymerization. The cargo‐free NPs were fabricated with the solvent evaporation method, and studies on NPs' characterizations were carried out. The hydrogen nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy spectra confirmed the synthesis of PHEMA‐S‐S‐PCL copolymer. Thermo‐gravimetric analysis curves indicated that the obtained PHEMA‐S‐S‐PCL copolymer had good thermostability. Transmission electron microscopy and dynamic light scatter results suggested that the cargo‐free NPs were in round shapes with an average diameter of 103.6 ± 0.12 nm. The low critical micelle concentration of cargo‐free NPs (7.9 × 10−4 mg/ml) indicated that these NPs would keep their spherical shapes after being attenuated by abundant liquid (e.g., blood or body fluid). Furthermore, these NPs showed high stability at the presence of bovine serum albumin. Therefore, it could be speculated that these NPs would not be absorbed by proteins in blood, and they could be used as a candidate carrier for drug delivery.

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Crystal structure of the disulfide-stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond

Cited by 17 publications

References 35 publications

Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges

Trapping a 96° domain rotation in two distinct conformations by engineered disulfide bridges

Three‐dimensional structures of carbohydrate determinants of Lewis system antigens: Implications for effective antibody targeting of cancer

Fabrication and characterization of glutathione‐responsive nanoparticles from the disulfide bond‐bridged block copolymer

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