Corrigendum: The Effects of Framework Mutations at the Variable Domain Interface on Antibody Affinity Maturation in an HIV-1 Broadly Neutralizing Antibody Lineage.

[This corrects the article DOI: 10.3389/fimmu.2020.01529.].

Identification and Characterization of a B-Raf Kinase α-Helix Critical for the Activity of MEK Kinase in MAPK Signaling.

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ∼3% of all cancers, and many drugs target the ATP binding site of the enzyme for its inhibition. Because B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an α-helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We assessed the binding between B-Raf mutants and MEK using pull downs and biolayer interferometry and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this α-helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf α-helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

The Effects of Framework Mutations at the Variable Domain Interface on Antibody Affinity Maturation in an HIV-1 Broadly Neutralizing Antibody Lineage.

Understanding affinity maturation of antibodies that can target many variants of HIV-1 is important for vaccine development. While the antigen-binding site of antibodies is known to mutate throughout the co-evolution of antibodies and viruses in infected individuals, the roles of the mutations in the antibody framework region are not well understood. Throughout affinity maturation, the CH103 broadly neutralizing antibody lineage, from an individual designated CH505, altered the orientation of one of its antibody variable domains. The change in orientation was a response to insertions in the variable loop 5 (V5) of the HIV envelope. In this study, we generated CH103 lineage antibody variants in which residues in the variable domain interface were mutated, and measured the binding to both autologous and heterologous HIV-1 envelopes. Our data show that very few mutations in an early intermediate antibody of the lineage can improve binding toward both autologous and heterologous HIV-1 envelopes. We also crystallized an antibody mutant to show that framework mutations alone can result in a shift in relative orientations of the variable domains. Taken together, our results demonstrate the functional importance of residues located outside the antigen-binding site in affinity maturation.

Structural Insights from HIV-Antibody Coevolution and Related Immunization Studies.

Human immunodeficiency virus type 1 (HIV-1) is a rapidly evolving pathogen that causes acquired immunodeficiency syndrome (AIDS) in humans. There are ∼30-35 million people infected with HIV around the world, and ∼25 million have died since the first reported cases in 1981. In addition, each year 2-3 million people become newly infected, and >1 million die of AIDS. An HIV-1 vaccine would help halt an AIDS pandemic, and efforts to develop a vaccine have focused on targeting the HIV-1 envelope, Env, found on the surface of the virus. A number of chronically infected individuals have been shown to produce antibodies, called broadly neutralizing antibodies (bnAbs), that target many strains of HIV-1 by binding to Env, thus suggesting promise for HIV-1 vaccine development. BnAbs take years to develop, and have a number of traits that inhibit their production; thus, a number of researchers are trying to understand the pathways that result in bnAb production, so that they can be elicited more rapidly by vaccination. This review discusses results and implications from two HIV-1-infected individuals studied longitudinally who produced bnAbs against two different sites on HIV-1 Env, and immunization studies that used Envs derived from those individuals.

HIV envelope V3 region mimic embodies key features of a broadly neutralizing antibody lineage epitope.

HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man9-V3") for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.