2·9 Å resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten

Axel T. Brünger; Daniel J. Leahy; Thomas R. Hynes; Robert O. Fox

doi:10.1016/0022-2836(91)80217-I

2·9 Å resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten

Axel T. Brünger, Daniel J. Leahy, Thomas R. Hynes, Robert O. Fox

Research output: Contribution to journal › Article › peer-review

101 Scopus citations

Abstract

The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2·9 Å resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (V_H, V_L, C_H1 and C_L) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was the refined 2·5 Å crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (γ1, κ, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19·5% for 12,129 unique reflections between 8·0 and 2·9 Å. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0·014 Å, and the r.m.s. deviation from ideal bond angles is 3·1°. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.

Original language	English (US)
Pages (from-to)	239-256
Number of pages	18
Journal	Journal of molecular biology
Volume	221
Issue number	1
DOIs	https://doi.org/10.1016/0022-2836(91)80217-I
State	Published - Sep 5 1991
Externally published	Yes

Keywords

PC-refinement
antibody
crystal structure
fab fragment
molecular replacement

ASJC Scopus subject areas

Structural Biology
Molecular Biology

Access to Document

10.1016/0022-2836(91)80217-I

Cite this

@article{7c02416b473946f697eaf536eff9cab7,

title = "2·9 {\AA} resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten",

abstract = "The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2·9 {\AA} resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was the refined 2·5 {\AA} crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (γ1, κ, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19·5% for 12,129 unique reflections between 8·0 and 2·9 {\AA}. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0·014 {\AA}, and the r.m.s. deviation from ideal bond angles is 3·1°. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.",

keywords = "PC-refinement, antibody, crystal structure, fab fragment, molecular replacement",

author = "Br{\"u}nger, {Axel T.} and Leahy, {Daniel J.} and Hynes, {Thomas R.} and Fox, {Robert O.}",

note = "Funding Information: This work was supported by the American Cancer Society (R.O.F., grant NP631), the Office of Naval Research (H. M. McConnell grant 1300014~90-J-1407), the NSF funded Pittsburgh Supercomputer Center (A.T.B.. grant DMB890008P). Calculations were also carried out on the CONVEX C-220 of t,he Yale Center for Structural Biology. We thank W. 4. Hendrickson, W. T. Weis and H. M. McConnell for helpful discussions and W. A. Hendrickson for generous use of the computing facilities at Columbia. We thank J. Johnson and M. Rossmann for access to the film processing facility at Purdue University and for helpful advice. D.J.L. is a fellow of the Helen Hay Whitney Foundation.",

year = "1991",

month = sep,

day = "5",

doi = "10.1016/0022-2836(91)80217-I",

language = "English (US)",

volume = "221",

pages = "239--256",

journal = "Journal of molecular biology",

issn = "0022-2836",

publisher = "Academic Press Inc.",

number = "1",

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TY - JOUR

T1 - 2·9 Å resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten

AU - Brünger, Axel T.

AU - Leahy, Daniel J.

AU - Hynes, Thomas R.

AU - Fox, Robert O.

N1 - Funding Information: This work was supported by the American Cancer Society (R.O.F., grant NP631), the Office of Naval Research (H. M. McConnell grant 1300014~90-J-1407), the NSF funded Pittsburgh Supercomputer Center (A.T.B.. grant DMB890008P). Calculations were also carried out on the CONVEX C-220 of t,he Yale Center for Structural Biology. We thank W. 4. Hendrickson, W. T. Weis and H. M. McConnell for helpful discussions and W. A. Hendrickson for generous use of the computing facilities at Columbia. We thank J. Johnson and M. Rossmann for access to the film processing facility at Purdue University and for helpful advice. D.J.L. is a fellow of the Helen Hay Whitney Foundation.

PY - 1991/9/5

Y1 - 1991/9/5

N2 - The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2·9 Å resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was the refined 2·5 Å crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (γ1, κ, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19·5% for 12,129 unique reflections between 8·0 and 2·9 Å. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0·014 Å, and the r.m.s. deviation from ideal bond angles is 3·1°. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.

AB - The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2·9 Å resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was the refined 2·5 Å crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (γ1, κ, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19·5% for 12,129 unique reflections between 8·0 and 2·9 Å. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0·014 Å, and the r.m.s. deviation from ideal bond angles is 3·1°. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.

KW - PC-refinement

KW - antibody

KW - crystal structure

KW - fab fragment

KW - molecular replacement

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U2 - 10.1016/0022-2836(91)80217-I

DO - 10.1016/0022-2836(91)80217-I

M3 - Article

C2 - 1920408

AN - SCOPUS:0025923239

SN - 0022-2836

VL - 221

SP - 239

EP - 256

JO - Journal of molecular biology

JF - Journal of molecular biology

IS - 1

ER -

2·9 Å resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten

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