Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding

ABSTRACT Most antibodies are multivalent, with the potential to bind with high avidity. However, neutralizing antibodies commonly bind to virions monovalently. Bivalent binding of a monoclonal antibody (MAb) to a virion has been documented only in a single case. Thus, the role of high avidity in antibody-mediated neutralization of viruses has not been defined clearly. In this study, we demonstrated that when an artificial 2F5 epitope was inserted in the gp120 V4 region so that an HIV-1 envelope glycoprotein (Env) trimer contains a natural 2F5 epitope in the gp41 membrane-proximal envelope region (MPER) and an artificially engineered 2F5 epitope in the gp120 V4 region, bivalent 2F5 IgG achieved greatly enhanced neutralization efficiency, with a 50% inhibitory concentration (IC50) decrease over a 2-log scale. In contrast, the monovalent 2F5 Fab fragment did not exhibit any appreciable change in neutralization efficiency in the same context. These results demonstrate that bivalent binding of 2F5 IgG to a single HIV-1 Env trimer results in dramatic enhancement of neutralization, probably through an increase in binding avidity. Furthermore, we demonstrated that bivalent binding of MAb 2F5 to the V4 region and MPER of an HIV-1 Env trimer can be achieved only in a specific configuration, providing an important insight into the structure of a native/infectious HIV-1 Env trimer. This specific binding configuration also establishes a useful standard that can be applied to evaluate the biological relevance of structural information on the HIV-1 Env trimer.

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Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v120.21.2880.2880 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2880-2880

Author(s):

Martin Trepel ◽

Fabian Muller ◽

Mareike Frick ◽

Janina Rahlff ◽

Claudia Wehr ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Phage Display ◽

B Cell ◽

Conflicts Of Interest ◽

Specific Binding ◽

Variable Region ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Fab Fragment

Abstract Abstract 2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods: The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results: Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence. Conclusions: These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients. Disclosures: No relevant conflicts of interest to declare.

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Crystal structure of a streptococcal protein G domain bound to an Fab fragment

Nature ◽

10.1038/359752a0 ◽

1992 ◽

Vol 359 (6397) ◽

pp. 752-754 ◽

Cited By ~ 129

Author(s):

Jeremy P. Derrick ◽

Dale B. Wigley

Keyword(s):

Crystal Structure ◽

Protein G ◽

Fab Fragment ◽

Streptococcal Protein G

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Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515737112 ◽

2015 ◽

Vol 112 (39) ◽

pp. E5391-E5400 ◽

Cited By ~ 98

Author(s):

Nikita Vasilyev ◽

Anna Polonskaia ◽

Jennifer C. Darnell ◽

Robert B. Darnell ◽

Dinshaw J. Patel ◽

...

Keyword(s):

Crystal Structure ◽

Rna Binding ◽

Specific Binding ◽

Fragile X ◽

Type I ◽

Shape Complementarity ◽

G Quadruplex ◽

Rna Duplexes ◽

Human Disorders

Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome (FXS) and autism. FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with guanine (G)-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. Here we report the 2.8-Å crystal structure of the complex between the human FMRP RGG peptide bound to the in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K+-stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex–quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation–π interactions, and multiple hydrogen bonds. Many of these interactions critically depend on a type I β-turn, a secondary structure element whose formation was not previously recognized in the RGG motif of FMRP. RNA mutagenesis and footprinting experiments indicate that interactions of the peptide with the duplex–quadruplex junction and the duplex of RNA are equally important for affinity and specificity of the RGG–RNA complex formation. These results suggest that specific binding of cellular RNAs by FMRP may involve hydrogen bonding with RNA duplexes and that RNA duplex recognition can be a characteristic RNA binding feature for RGG motifs in other proteins.

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