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1.
Proteins ; 91(9): 1316-1328, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37376973

RESUMEN

In the last years, antibodies have emerged as a promising new class of therapeutics, due to their combination of high specificity with long serum half-life and low risk of side-effects. Diabodies are a popular novel antibody format, consisting of two Fv domains connected with short linkers. Like IgG antibodies, they simultaneously bind two target proteins. However, they offer altered properties, given their smaller size and higher rigidity. In this study, we conducted the-to our knowledge-first molecular dynamics (MD) simulations of diabodies and find a surprisingly high conformational flexibility in the relative orientation of the two Fv domains. We observe rigidifying effects through the introduction of disulfide bonds in the Fv -Fv interface and characterize the effect of different disulfide bond locations on the conformation. Additionally, we compare VH -VL orientations and paratope dynamics between diabodies and an antigen binding fragment (Fab) of the same sequence. We find mostly consistent structures and dynamics, indicating similar antigen binding properties. The most significant differences can be found within the CDR-H2 loop dynamics. Of all CDR loops, the CDR-H2 is located closest to the artificial Fv -Fv interface. All examined diabodies show similar VH -VL orientations, Fv -Fv packing and CDR loop conformations. However, the variant with a P14C-K64C disulfide bond differs most from the Fab in our measures, including the CDR-H3 loop conformational ensemble. This suggests altered antigen binding properties and underlines the need for careful validation of the disulfide bond locations in diabodies.


Asunto(s)
Anticuerpos , Fragmentos Fab de Inmunoglobulinas , Conformación Proteica , Sitios de Unión de Anticuerpos , Fragmentos Fab de Inmunoglobulinas/química , Disulfuros
2.
MAbs ; 12(1): 1744328, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32264741

RESUMEN

In the past decade, the relevance of antibodies as therapeutics has increased substantially. Therefore, structural and functional characterization, in particular of the complementarity-determining regions (CDRs), is crucial to the design and engineering of antibodies with unique binding properties. Various studies have focused on classifying the CDR loops into a small set of main-chain conformations to facilitate antibody design by assuming that certain sequences can only adopt a limited number of conformations. Here, we present a kinetic classification of CDR loop structures as ensembles in solution. Using molecular dynamics simulations in combination with strong experimental structural information, we observe conformational transitions between canonical clusters and additional dominant solution structures in the micro-to-millisecond timescale for all CDR loops, independent of length and sequence composition. Besides identifying all relevant conformations in solution, our results revealed that various canonical cluster medians actually belong to the same kinetic minimum. Additionally, we reconstruct the kinetics and probabilities of the conformational transitions between canonical clusters, and thereby extend the model of static canonical structures to reveal a dynamic conformational ensemble in solution as a new paradigm in the field of antibody structure design.Abbreviations: CDR: Complementary-determining region; Fv: Antibody variable fragment; PCCA: Perron cluster analysis; tICA: Time-lagged independent component analysis; VH: Heavy chain variable region; VL: Light chain variable region.


Asunto(s)
Regiones Determinantes de Complementariedad/química , Conformación Proteica , Animales , Cristalografía por Rayos X/métodos , Humanos , Simulación de Dinámica Molecular
3.
Commun Biol ; 3(1): 589, 2020 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-33082531

RESUMEN

In the last decades, antibodies have emerged as one of the most important and successful classes of biopharmaceuticals. The highest variability and diversity of an antibody is concentrated on six hypervariable loops, also known as complementarity determining regions (CDRs) shaping the antigen-binding site, the paratope. Whereas it was assumed that certain sequences can only adopt a limited set of backbone conformations, in this study we present a kinetic classification of several paratope states in solution. Using molecular dynamics simulations in combination with experimental structural information we capture the involved conformational transitions between different canonical clusters and additional dominant solution structures occurring in the micro-to-millisecond timescale. Furthermore, we observe a strong correlation of CDR loop movements. Another important aspect when characterizing different paratope states is the relative VH/VL orientation and the influence of the distinct CDR loop states on the VH/VL interface. Conformational rearrangements of the CDR loops do not only have an effect on the relative VH/VL orientations, but also influence in some cases the elbow-angle dynamics and shift the respective distributions. Thus, our results show that antibodies exist as several interconverting paratope states, each contributing to the antibody's properties.


Asunto(s)
Anticuerpos/química , Sitios de Unión de Anticuerpos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Dominios y Motivos de Interacción de Proteínas , Anticuerpos/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica
4.
Front Immunol ; 10: 2652, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31803187

RESUMEN

Sequence and structural diversity of antibodies are concentrated on six hypervariable loops, also known as the complementarity determining regions (CDRs). Five of six antibody CDR loops presumably adopt a so-called canonical structure out of a limited number of conformations. However, here we show for four antibody CDR-L3 loops differing in length and sequence, that each loop undergoes conformational transitions between different canonical structures. By extensive sampling in combination with Markov-state models we reconstruct the kinetics and probabilities of the transitions between canonical structures. Additionally, for these four CDR-L3 loops, we identify all relevant conformations in solution. Thereby we extend the model of static canonical structures to a dynamic conformational ensemble as a new paradigm in the field of antibody structure design.


Asunto(s)
Regiones Determinantes de Complementariedad/química , Anticuerpos/química , Modelos Moleculares , Conformación Proteica
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