Kinetic epitope mapping of the chicken lysozyme.HyHEL‐10 fab complex: Delineation of docking trajectories

We measured the influence of 14 mutations and 5 environmental variables (buffer perturbation) on the association and dissociation rate of a camel single domain antibody (cAb-Lys3) interacting with hen egg white lysozyme using a surface plasmon resonance-based biosensor. Based on this data set, we constructed quantitative predictive models for both kinetic (k a and k d ) constants as for the affinity constant (K d ). Mutations, after parameterization by quantitative descriptors, and buffers were selected using multivariate experimental design. These models were able to predict the corresponding parameters of four new variants of cAb-Lys3. Moreover, the models provide insights to the important chemical aspects of the interacting residues, which are difficult to deduce from the crystal structure. Our approach provides useful physicochemical information of protein-protein interactions in general. The information obtained from this kind of analysis complements and goes beyond that of conventional methods like alanine scanning and substitution by closely related amino acids. The mathematical modeling may contribute to a rational approach in the optimization of bio-molecules of biotechnological interest.Protein-protein interactions attract a lot of attention in this era of proteomics and antibody technology. Naive and synthetic antibody libraries in combination with phage and ribosome display are now routinely used to obtain antibody molecules with desired properties and specificities (1-6). However, the affinity and/or stability of the original binder retrieved from the library often requires improvements. Molecular evolution techniques (1-8) and rational design using sophisticated computer programs (9 -16) are two approaches to achieve this goal. Drawbacks to the former strategy are its dependence on the generation of large libraries, the difficulty in discrimination between binders with small differences in affinity, and the bias in favor of binders with improved expression levels (1,3). Additionally, codon bias and the generation of nonfunctional or deleterious mutants are major drawbacks of this strategy (1, 3). The rational design strategy largely depends on the availability of the structure of the protein-protein complex, and, although predictive algorithms have been improved significantly over the years, predictions of interaction energies, especially those related to amino acid modifications, remain highly problematic (9 -16).It is generally accepted that experimental data on the energetic contribution of the modified amino acids are required to develop algorithms relating binding energetics to particular amino acid modifications. Many analyses have used site-directed mutagenesis, especially alanine scanning (17-21) and substitution by closely related amino acids (21-23), to determine the energetic contributions of the amino acid side chains and functional groups. These strategies have shown to be very attractive because of their simplicity. However, they often lead to misinterpretations and inconclusive resul...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Kinetic and Affinity Predictions of a Protein-Protein Interaction Using Multivariate Experimental Design

Genst¹,

Areskoug²,

Decanniere³

et al. 2002

“…In addition, both antibody fragments eliminate the HEWL-mediated lysis (29) of Micrococcus luteus cells. Furthermore, Biebrich Scarlett, a dye interacting with the carbohydrate substrate D, E, and F subsites of HEWL (30), competitively inhibited both VHH-HEWL associations (not shown).…”

Section: Ontogeny Of D2-l19 and Cab-lys2-twomentioning

confidence: 99%

Strong in Vivo Maturation Compensates for Structurally Restricted H3 Loops in Antibody Repertoires

Genst

Silence

Ghahroudi

et al. 2005

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires.The interaction of conventional antibodies with antigens is mediated by up to six dedicated hypervariable loops, three (H1-H3) 1 in the variable domain of the heavy chain (VH) and three (L1-L3) in the variable domain of the light chain (VL) (1). Within the antigen-combining site, the H3 loop is the major determinant of antibody diversity and the major contributor for overall antigen affinity and specificity (1-3). This loop is generated after recombining one V, one D, and one J gene out of a pool. Imprecision in the V(D)J recombination event, with concomitant nucleotide deletions and N or P nucleotide additions (4, 5), makes it possible for a particular D gene to occur in different locations and reading frames within the H3-encoded region (4, 5). Hence, the sequence of the H3 loop becomes the most diverse of all antigen binding loops.The crystal structure determination of different antibodies revealed the presence of canonical structures for all antigen binding loops (L1-L3, H1, and H2) except for the H3 loop (6, 7). The folding of a particular loop into a canonical structure is dictated by a small number of conserved key residues within the hypervariable sequences. The immense sequence diversity of the H3 loop with the D-encoded part, which can be located anywhere within the loop, obviously precludes an easy assignment of such key residues. Moreover, because the number of possible V(D)J recombinations exceeds by far the number of crystal structures that are available, it is as yet impossible to investigate whether the D-encoded part of the loop adopts different or similar backbone architectures when present in a different V-J surrounding. Likewise, it remains an open question whether the same epitope on a large antigen will be recognized by antibodies originating from differ...

“…In typical stopped-flow binding measurements, the protein and ligand are rapidly mixed, and the extent of the reaction is monitored as a function of time using techniques including spectroscopic absorbance (21), fluorescence (10,26,27), enzymatic activity (1,28,29), and surface plasmon resonance (30,31). These techniques are limited by the requirement that the lifetimes of the free and bound states must be severalfold longer than the time required to completely mix the protein and ligand solutions; otherwise, the reaction proceeds nearly to completion before the start of the measurement period.…”

mentioning

confidence: 99%

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

Meneses

Mittermaier

2014

Background: Electrostatic interactions are known accelerate binding in protein complexes with long lifetimes (minutes to hours). Results: NMR and calorimetry were used to characterize binding kinetics in short lived (ϳ1 ms) protein-peptide complexes. Conclusion: Electrostatic enhancement is much less and basal (electrostatic-free) association rates are greater than previously observed. Significance: Electrostatics may play different roles in short lived and long lived protein complexes.