Rational engineering of a human anti-dengue antibody through experimentally validated computational docking.

Antibodies play an increasing pivotal role in both basic research and the biopharmaceutical sector, therefore technology for characterizing and improving their properties through rational engineering is desirable. This is a difficult task thought to require high-resolution x-ray structures, which are not always available. We, instead, use a combination of solution NMR epitope mapping and computational docking to investigate the structure of a human antibody in complex with the four Dengue virus serotypes. Analysis of the resulting models allows us to design several antibody mutants altering its properties in a predictable manner, changing its binding selectivity and ultimately improving its ability to neutralize the virus by up to 40 fold. The successful rational design of antibody mutants is a testament to the accuracy achievable by combining experimental NMR epitope mapping with computational docking and to the possibility of applying it to study antibody/pathogen interactions.

HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4.

After tissue damage, inflammatory cells infiltrate the tissue and release proinflammatory cytokines. HMGB1 (high mobility group box 1), a nuclear protein released by necrotic and severely stressed cells, promotes cytokine release via its interaction with the TLR4 (Toll-like receptor 4) receptor and cell migration via an unknown mechanism. We show that HMGB1-induced recruitment of inflammatory cells depends on CXCL12. HMGB1 and CXCL12 form a heterocomplex, which we characterized by nuclear magnetic resonance and surface plasmon resonance, that acts exclusively through CXCR4 and not through other HMGB1 receptors. Fluorescence resonance energy transfer data show that the HMGB1-CXCL12 heterocomplex promotes different conformational rearrangements of CXCR4 from that of CXCL12 alone. Mononuclear cell recruitment in vivo into air pouches and injured muscles depends on the heterocomplex and is inhibited by AMD3100 and glycyrrhizin. Thus, inflammatory cell recruitment and activation both depend on HMGB1 via different mechanisms.

Computational docking of antibody-antigen complexes, opportunities and pitfalls illustrated by influenza hemagglutinin.

Antibodies play an increasingly important role in both basic research and the pharmaceutical industry. Since their efficiency depends, in ultimate analysis, on their atomic interactions with an antigen, studying such interactions is important to understand how they function and, in the long run, to design new molecules with desired properties. Computational docking, the process of predicting the conformation of a complex from its separated components, is emerging as a fast and affordable technique for the structural characterization of antibody-antigen complexes. In this manuscript, we first describe the different computational strategies for the modeling of antibodies and docking of their complexes, and then predict the binding of two antibodies to the stalk region of influenza hemagglutinin, an important pharmaceutical target. The purpose is two-fold: on a general note, we want to illustrate the advantages and pitfalls of computational docking with a practical example, using different approaches and comparing the results to known experimental structures. On a more specific note, we want to assess if docking can be successful in characterizing the binding to the same influenza epitope of other antibodies with unknown structure, which has practical relevance for pharmaceutical and biological research. The paper clearly shows that some of the computational docking predictions can be very accurate, but the algorithm often fails to discriminate them from inaccurate solutions. It is of paramount importance, therefore, to use rapidly obtained experimental data to validate the computational results.

A blue light-inducible phosphodiesterase activity in the cyanobacterium Synechococcus elongatus.

A blue light-inducible phosphodiesterase (PDE) activity, specific for the hydrolysis of cyclic di-GMP (c-di-GMP), has been identified in a recombinant protein from Synechococcus elongatus. Blue light (BL) activation is accomplished by a light, oxygen, voltage (LOV) domain, found in plant phototropins and bacterial BL photoreceptors. The genome of S. elongatus contains two genes coding for proteins with LOV domains fused to EAL domains (SL1 and SL2). In both cases, a GGDEF motif is placed in between the LOV and the EAL motifs. Such arrangement is frequently found with diguanylate-cyclase (DGC) functions that form c-di-GMP. Cyclic di-GMP acts as a second messenger molecule regulating biofilm formation in many microbial species. Both enzyme activities modulate the intracellular level of this second messenger, although in most proteins only one of the two enzyme functions is active. Both S. elongatus LOV-GGDEF-EAL proteins were expressed in full length or as truncated proteins. Only the SL2 protein, expressed as a LOV-GGDEF-EAL construct, showed an increase of PDE activity upon BL irradiation, demonstrating this activity for the first time in a LOV-domain protein. Addition of GTP or c-di-GMP did not affect the observed enzymatic activity. In none of the full-length or truncated proteins was a DGC activity detected.

Interdomain signalling in the blue-light sensing and GTP-binding protein YtvA: a mutagenesis study uncovering the importance of specific protein sites.

YtvA from Bacillus subtilis is a blue-light responsive, flavin-binding photoreceptor, built of a light-sensing LOV domain (aa 25-126) and an NTP (nucleoside triphosphate)-binding STAS domain (aa 147-261). The STAS domain is supposed to be the effector part of the protein or a secondary switch. Both domains are connected by a linker polypeptide. The active form of YtvA is generated upon light excitation, causing the formation of a covalent bond between a cysteine residue (Cys62) in the LOV domain and the position 4a of the flavin chromophore. This photoadduct formation within the LOV domain results in a conformational change of the NTP-binding cavity, evidencing intra-protein signal transmission. We have previously shown that Glu105, localized on the beta-scaffold of the LOV-core, is involved in this process. Here, we extend this work by the identification of further residues that upon mutation supress or strongly impair signal transmission by interfering with the communication between the two domains. These comprise L106 and D109 on the LOV domain; K130 and K134 on the linker region; D193, L194 and G196 within the DLSG GTP-binding motif (switch region) and N201 on the STAS domain. Furthermore in the mutated S195A and D193A proteins, GTP affinity is diminished. Other mutations investigated have little or no effect on signal transmission and GTP-binding affinity: R63K that was found to accelerate the thermal recovery of the parent state ca. ten-fold; K128A, Q129A and Y132A within the linker region, and S183A and S212A on the STAS domain. The results show a key role of the LOV domain beta-scaffold and of positively charged residues within the linker for intra-protein signal transmission. Furthermore they evidence the conformational switch function of a structurally conserved strand-loop-helix region (bearing the DLSG GTP-binding motif and N201) within the STAS domain that constitutes a novel GTP-binding fold.