6NEX

Fab fragment of anti-cocaine antibody h2E2

PDB DOI: https://doi.org/10.2210/pdb6NEX/pdb

Classification: IMMUNE SYSTEM
Organism(s): Mus musculus
Expression System: Cricetulus griseus
Mutation(s): No

Deposited: 2018-12-18 Released: 2019-11-20
Deposition Author(s): Pokkuluri, P.R., Tan, K.

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.15 Å
R-Value Free: 0.254
R-Value Work: 0.214
R-Value Observed: 0.216

wwPDB Validation 3D Report Full Report

This is version 2.1 of the entry. See complete history.

Literature

Structural analysis of free and liganded forms of the Fab fragment of a high-affinity anti-cocaine antibody, h2E2.

Tan, K., Zhou, M., Ahrendt, A.J., Duke, N.E.C., Tabaja, N., Ball, W.J., Kirley, T.L., Norman, A.B., Joachimiak, A., Schiffer, M., Wilton, R., Pokkuluri, P.R.

(2019) Acta Crystallogr F Struct Biol Commun 75: 697-706

PubMed: 31702583 Search on PubMedSearch on PubMed Central
DOI: https://doi.org/10.1107/S2053230X19013608
Primary Citation of Related Structures:
6NEX, 6NFN

PubMed Abstract:
A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab-benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.

Organizational Affiliation

Structural Biology Center, X-ray Science Division, Argonne National Laboratory, Lemont, IL 60439, USA.

Macromolecule Content

Total Structure Weight: 92.64 kDa
Atom Count: 6,486
Modelled Residue Count: 851
Deposited Residue Count: 866
Unique protein chains: 4

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
Anitgen binding fragment light chain	A [auth L]	215	Mus musculus	Mutation(s): 0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations Expand
Reference Sequence

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Entity ID: 2
Molecule	Chains	Sequence Length	Organism	Details	Image
Antigen binding fragment heavy chain	B [auth H]	218	Mus musculus	Mutation(s): 0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations Expand
Reference Sequence

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(by identity cutoff) | 3D Structure

Entity ID: 3
Molecule	Chains	Sequence Length	Organism	Details	Image
Anitgen binding fragment light chain	C [auth A]	215	Mus musculus	Mutation(s): 0
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations Expand
Reference Sequence

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(by identity cutoff) | 3D Structure

Entity ID: 4
Molecule	Chains	Sequence Length	Organism	Details	Image
Ighg protein	D [auth B]	218	Mus musculus	Mutation(s): 0 Gene Names: Ighg, AU044919
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 3 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
ACT Query on ACT Download Ideal Coordinates CCD File SDF format, chain J [auth H] MOL2 format, chain J [auth H]	J [auth H]	ACETATE ION C₂ H₃ O₂ QTBSBXVTEAMEQO-UHFFFAOYSA-M		Interactions Focus chain J [auth H] Interactions & Density Focus chain J [auth H]
FMT Query on FMT Download Ideal Coordinates CCD File SDF format, chain G [auth L] SDF format, chain H [auth L] SDF format, chain I [auth L] SDF format, chain L [auth A] MOL2 format, chain G [auth L] MOL2 format, chain H [auth L] MOL2 format, chain I [auth L] MOL2 format, chain L [auth A]	G [auth L], H [auth L], I [auth L], L [auth A]	FORMIC ACID C H₂ O₂ BDAGIHXWWSANSR-UHFFFAOYSA-N		Interactions Focus chain G [auth L] Focus chain H [auth L] Focus chain I [auth L] Focus chain L [auth A] Interactions & Density Focus chain G [auth L] Focus chain H [auth L] Focus chain I [auth L] Focus chain L [auth A]
CL Query on CL Download Ideal Coordinates CCD File SDF format, chain E [auth L] SDF format, chain F [auth L] SDF format, chain K [auth A] MOL2 format, chain E [auth L] MOL2 format, chain F [auth L] MOL2 format, chain K [auth A]	E [auth L], F [auth L], K [auth A]	CHLORIDE ION Cl VEXZGXHMUGYJMC-UHFFFAOYSA-M		Interactions Focus chain E [auth L] Focus chain F [auth L] Focus chain K [auth A] Interactions & Density Focus chain E [auth L] Focus chain F [auth L] Focus chain K [auth A]

Modified Residues 2 Unique
ID	Chains	Type	Formula	2D Diagram	Parent
MLY Query on MLY	A [auth L]	L-PEPTIDE LINKING	C₈ H₁₈ N₂ O₂		LYS
PCA Query on PCA	A [auth L]	L-PEPTIDE LINKING	C₅ H₇ N O₃		GLN

Experimental Data & Validation

Experimental Data

Method: X-RAY DIFFRACTION
Resolution: 2.15 Å
R-Value Free: 0.254
R-Value Work: 0.214
R-Value Observed: 0.216
Space Group: C 1 2 1

Unit Cell:

Length ( Å )	Angle ( ˚ )
a = 110.787	α = 90
b = 128.435	β = 125.258
c = 91.196	γ = 90

Software Package:

Software Name	Purpose
SBC-Collect	data collection
PHENIX	refinement
HKL-3000	data reduction
HKL-3000	data scaling
PHASER	phasing

Structure Validation

View Full Validation Report

Entry History

Deposition Data

Released Date: 2019-11-20

Deposition Author(s):

Pokkuluri, P.R.

Tan, K.

Revision History (Full details and data files)

Version 1.0: 2019-11-20
Type: Initial release
Version 2.0: 2020-01-22
Changes: Polymer sequence, Structure summary
Version 2.1: 2024-04-03
Changes: Data collection, Database references, Refinement description