Biomedical subjects

Diego U Ferreiro

Publications and source records attributed to Diego U Ferreiro.

7 recordsLinked to original sources

PubMed2006

Antibody recognition of a flexible epitope at the DNA binding site of the human papillomavirus transcriptional regulator E2.

We have obtained a monoclonal antibody (ED15) against the C-terminal DNA-binding domain of the high-risk human papillomavirus strain-16 E2 protein that strongly interferes with its DNA-binding activity. We here characterize the recognition mechanism of this antibody and find that the ED15-E2 interaction has a strong electrostatic component, which correlates with the high proportion of acidic residues found in the antibody combining site. Further circular dichroism experiments in the presence of phosphate show that, in addition to electrostatic screening of key potential interactions, ionic strength affects the conformation of the epitope. In addition, the interaction is strongly modulated by pH, which correlates with the local flexibility of the epitope rather than the presence of pH sensitive residues at the interface. Noticeably, this finding is well correlated with the strong entropic component of the interaction. Site directed mutagenesis indicates that the ED15 epitope involves at least part of the DNA-binding helix of E2, explaining the mAb inhibitory activity. At physiological salt concentrations, the equilibrium dissociation constant of the E2-ED15 interaction is 10(-7) M and the association rate is 10(4) M-1 s-1, at least 1 order of magnitude slower than those generally reported in the most extensively described "nonflexible" antibody-protein interactions, indicating the presence of a slow conformational rearrangement on the antigen as the rate-limiting step. The crucial role of antigen flexibility in antibody-protein recognition is discussed.

Amino Acid Sequence↗

PubMed2006

Stabilizing IkappaBalpha by "consensus" design.

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

Amino Acid Sequence↗

PubMed2005

The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family.

Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.

Algorithms↗

PubMed2005

The folding mechanism of a dimeric beta-barrel domain.

The dimeric beta-barrel domain is an unusual topology, shared only by two viral origin binding proteins, where secondary, tertiary and quaternary structure are coupled, and where the dimerization interface is composed of two four-stranded half-beta-barrels. The folding of the DNA binding domain of the E2 transcriptional regulator from human papillomavirus, strain-16, takes place through a stable and compact monomeric intermediate, with 31% the stability of the folded dimeric domain. Double jump multiple wavelength experiments allowed the reconstruction of the fluorescence spectrum of the monomeric intermediate at 100 milliseconds, indicating that tryptophan residues, otherwise buried in the folded state, are accessible to the solvent. Burial of surface area as well as differential behavior to ionic strength and pH with respect to the native ground state, plus the impossibility of having over 2500 A2 of surface area of the half-barrel exposed to the solvent, indicates that the formation of a non-native compact tertiary structure precedes the assembly of native quaternary structure. The monomeric intermediate can dimerize, albeit with a weaker affinity (approximately 1 microM), to yield a non-native dimeric intermediate, which rearranges to the native dimer through a parallel folding channel, with a unimolecular rate-limiting step. Folding pathways from either acid or urea unfolded states are identical, making the folding model robust. Unfolding takes place through a major phase accounting for apparently all the secondary structure change, with identical rate constant to that of the fluorescence unfolding experiment. In contrast to the folding direction, no unfolding intermediate was found.

Dimerization↗

PubMed2005

Free energy contributions to direct readout of a DNA sequence.

The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.

Amino Acid Sequence↗

PubMed2003

A protein-DNA binding mechanism proceeds through multi-state or two-state parallel pathways.

The DNA-binding mechanism of the dimeric C-terminal domain of the papillomavirus E2 protein with its specific DNA target was investigated and shown to proceed through two parallel pathways. A sequential multi-step reaction is initiated by the diffusion-controlled formation of an encounter complex, with no evidence of base sequence discrimination capacity. Following a substantial conformational rearrangement of the protein, a solvent exclusion step leading to the formation of a final protein-DNA complex was identified. This last step involves the largest burial of surface area from the interface and involves the consolidation of the direct readout of the DNA bases. Double-jump stopped-flow experiments allowed us to characterize the sequence of events and demonstrated that a fast-formed consolidated complex can take place through a parallel route. We present the simplest model for the overall mechanism with a description of all the intermediate species in energetic terms.

Base Sequence↗

PubMed2003

Specific antibody-DNA interaction: a novel strategy for tight DNA recognition.

Anti-double-stranded DNA monoclonal antibodies against a viral transcriptional regulatory site are capable of discriminating single-base replacements with affinities of 1 x 10(-)(9) M, which were optimized for the length of the duplex used as the immunogen. Their affinity for DNA duplexes of increasing length is lower, but reaches a plateau at 2 x 10(-)(8) M, still a fairly high affinity compared to those of most known natural anti-DNA antibodies. The ability of the antibodies to bind to a 166 bp DNA fragment containing the specific sequence strongly suggests that these have the potential of binding the specific sequence within larger genomic DNA fragments. Electrostatic interactions do not play a significant role, the opposite of what is observed in natural DNA binding interfaces. In addition, the insensitivity of the antibody-DNA interaction to solute effects is indicative of a marginal participation of water molecules at the interface compared to the level of participation at the natural E2-DNA interface. Spectroscopic evidence of base unstacking strongly suggests substantial denaturation of antibody-bound DNA, in agreement with thermodynamic results that show an unusual positive heat capacity change, which could be explained at least in part by the exposure of DNA bases upon binding. Lower local DNA stability cooperates with sequence recognition in producing the highest binding affinity. A slow rate of antibody-DNA association indicates an energy barrier imposed by conformational rearrangements, as opposed to an electrostatically assisted diffusion-controlled collision in the E2 DNA binding domain. While the E2-DNA interaction takes place through a typical direct readout mechanism, the anti-double-stranded DNA monoclonal antibody-DNA interaction could be viewed as a distinctive case of indirect readout with a significant distortion in the DNA conformation. However, the precise mechanism with which the DNA bases are accommodated in the antibody combining site will require structural analysis at atomic resolution. These results constitute a first stage for unveiling the unusual molecular recognition mechanism of a specific DNA sequence by antibodies. This mechanism could represent the strategy with which the immune system tightly and specifically recognizes a DNA antigen.

Animals↗