Energetic analysis of an antigen/antibody interface: alanine scanning mutagenesis and double mutant cycles on the HyHEL-10/lysozyme interaction.


Abstract

Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-10/HEWL complex demonstrates that the energetically important side chains (hot spots) of both partners are in contact. A plot of deltadeltaG(HyHEL-10_mutant) vs. deltadeltaG(HEWL_mutant) for the five of six interacting side-chain hydrogen bonds is linear (Slope = 1). Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32(H)N/K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the deltadeltaG values of the Tyr --> Phe vs. the corresponding Tyr --> Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total deltadeltaG observed for the Tyr --> Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31D(L)/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction, deltadeltaG(int), obtained from double mutant cycle analysis showed that deltadeltaG(int) for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large deltadeltaG values are analyzed.

Submission Details

ID: QwUHF3gh

Submitter: Shu-Ching Ou

Submission Date: Feb. 25, 2019, 1:03 p.m.

Version: 1

Publication Details
Pons J;Rajpal A;Kirsch JF,Protein Sci (1999) Energetic analysis of an antigen/antibody interface: alanine scanning mutagenesis and double mutant cycles on the HyHEL-10/lysozyme interaction. PMID:10338006
Additional Information

In the original study, position 95 on wild-type HYHEL-10 IGG1 Fab heavy chain is W instead of C in the pdb (and the AB-Bind article).

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2IHL 1993-06-29T00:00:00+0000 1.4 LYSOZYME (E.C.3.2.1.17) (JAPANESE QUAIL)
1FBI 1995-01-19T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF A CROSS-REACTION COMPLEX BETWEEN FAB F9.13.7 AND GUINEA-FOWL LYSOZYME
1GHL 1993-05-04T00:00:00+0000 2.1 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1JHL 1993-05-04T00:00:00+0000 2.4 THREE-DIMENSIONAL STRUCTURE OF A HETEROCLITIC ANTIGEN-ANTIBODY CROSS-REACTION COMPLEX
1HHL 1993-05-04T00:00:00+0000 1.9 THE THREE-DIMENSIONAL STRUCTURE OF PHEASANT AND GUINEA-FOWL EGG LYSOZYMES
1BQL 1995-02-03T00:00:00+0000 2.6 STRUCTURE OF AN ANTI-HEL FAB FRAGMENT COMPLEXED WITH BOBWHITE QUAIL LYSOZYME
1DKK 1996-01-10T00:00:00+0000 1.9 BOBWHITE QUAIL LYSOZYME WITH NITRATE
1DKJ 1996-01-10T00:00:00+0000 2.0 BOBWHITE QUAIL LYSOZYME
1JTP 2001-08-21T00:00:00+0000 1.9 Degenerate interfaces in antigen-antibody complexes
3LZ2 1991-09-13T00:00:00+0000 2.5 STRUCTURE DETERMINATION OF TURKEY EGG WHITE LYSOZYME USING LAUE DIFFRACTION

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 L HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P01837 IGKC_MOUSE
100.0 H HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P01868 IGHG1_MOUSE
100.0 H HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P01869 IGH1M_MOUSE
92.2 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P49663 LYSC_PHAVE
93.0 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P81711 LYSC_SYRSO
93.0 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P00702 LYSC_PHACO
94.6 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P24533 LYSC_SYRRE
93.0 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P24364 LYSC_LOPLE
92.2 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P00704 LYSC_NUMME
94.6 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P00703 LYSC_MELGA
95.3 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C Q7LZT2 LYSC_TRATE
95.3 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P22910 LYSC_CHRAM
96.1 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P19849 LYSC_PAVCR
95.3 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P00701 LYSC_COTJA
96.1 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C Q7LZI3 LYSC_TRASA
96.9 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C Q7LZP9 LYSC_LOPIM
96.9 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C Q7LZQ0 LYSC_CATWA
96.9 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P00699 LYSC_CALCC
96.9 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P00700 LYSC_COLVI
100.0 Y HYHEL-10 IGG1 FAB (HEAVY CHAIN),HYHEL-10 IGG1 FAB (LIGHT CHAIN),Lysozyme C P00698 LYSC_CHICK