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Structural features of interfacial tyrosine residue in ROBO1 fibronectin domain-antibody complex: Crystallographic, thermodynamic, and molecular dynamic analyses.

ABSTRACT: ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the antibody using molecular dynamics (MD) and thermodynamics. The structural comparison of B2212A Fab between the complex and the free form revealed that the interfacial Tyr(L) 50 (superscripts L, H, and F stand for the residues in the light chain, heavy chain, and Fn3, respectively) played important roles in Fn3 recognition. That is, the aromatic ring of Tyr(L) 50 pivoted toward Phe(F) 68, forming a CH/? interaction and a new hydrogen bond with the carbonyl O atom of Phe(F) 68. MD simulations predicted that the Tyr(L) 50-Phe(F) 68 interaction almost entirely dominated Fab-Fn3 binding, and Ala-substitution of Tyr(L) 50 led to a reduced binding of the resultant complex. On the contrary, isothermal titration calorimetry experiments underscored that Ala-substitution of Tyr(L) 50 caused an increase of the binding enthalpy between B2212A and Fn3, but importantly, it induced an increase of the binding entropy, resulting in a suppression of loss in the Gibbs free energy in total. These results suggest that mutation analysis considering the binding entropy as well as the binding enthalpy will aid in the development of novel antibody drugs for hepatocellular carcinoma.

SUBMITTER: Nakayama T

PROVIDER: S-EPMC4353359 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Structural features of interfacial tyrosine residue in ROBO1 fibronectin domain-antibody complex: Crystallographic, thermodynamic, and molecular dynamic analyses.

Nakayama Taisuke T Mizohata Eiichi E Yamashita Takefumi T Nagatoishi Satoru S Nakakido Makoto M Iwanari Hiroko H Mochizuki Yasuhiro Y Kado Yuji Y Yokota Yuki Y Satoh Reiko R Tsumoto Kouhei K Fujitani Hideaki H Kodama Tatsuhiko T Hamakubo Takao T Inoue Tsuyoshi T

Protein science : a publication of the Protein Society 20150113 3

ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the an ...[more]

PMID: 25492858

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Project description:The formation and crystal structure of the binary Cu3As phase have been re-investigated. Some physical properties were then measured on both single crystal and polycrystalline bulk. Cu3As melts congruently at 835 °C. At room temperature (RT), this compound has been found to crystallize in the hexagonal Cu3P prototype (hP24, P63cm) with lattice parameters: a = 7.1393(1) Å and c = 7.3113(1) Å, rather than in the anti HoH3-type (hP24, P-3c1) as indicated in literature. A small compositional range of 74.0-75.5 at.% Cu (26.0-24.5 at.% As) was found for samples synthesized at 300 and 400 °C; a corresponding slight understoichiometry is found in one out of the four Cu atomic sites, leading to the final refined composition Cu2.882(1)As. The present results disprove a change in the crystal structure above RT actually reported in the phase diagram (from γ' to γ on heating). Instead, below RT, at T = 243 K (-30 °C), a first-order structural transition to a trigonal low-temperature superstructure, LT-Cu3-xAs (hP72, P-3c1) has been found. The LT polymorph is metrically related to the RT one, having the c lattice parameter three times larger: a = 7.110(2) Å and c = 21.879(4) Å. Both the high- and low-temperature polymorphs are characterized by the presence of a tridimensional (3D) uncommon and rigid Cu sublattice of the lonsdaleite type (Cu atoms tetrahedrally bonded), which remains almost unaffected by the structural change(s), and characteristic layers of triangular 'Cu3As'-units (each hosting one As atom at the center, interconnected each other by sharing the three vertices). The first-order transition is then followed by an additional structural change when lowering the temperature, which induces doubling of also the lattice parameter a. Differential scanning calorimetry nicely detects the first low-temperature structural change occurring at T = 243 K, with an associated enthalpy difference, ΔH(TR), of approximately 2 J/g (0.53 kJ/mol). Low-temperature electrical resistivity shows a typical metallic behavior; clear anomalies are detected in correspondence to the solid-state transformations. The Seebeck coefficient, measured as a function of temperature, highlights a conduction of n-type. The temperature dependence of the magnetic susceptibility displays an overall constant diamagnetic response.

Dataset Information

Structural features of interfacial tyrosine residue in ROBO1 fibronectin domain-antibody complex: Crystallographic, thermodynamic, and molecular dynamic analyses.

Publications

Structural features of interfacial tyrosine residue in ROBO1 fibronectin domain-antibody complex: Crystallographic, thermodynamic, and molecular dynamic analyses.

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