Transcription factor NF-κB is modulated by symbiotic status in a sea anemone model of cnidarian bleaching.

Transcription factor NF-κB plays a central role in immunity from fruit flies to humans, and NF-κB activity is altered in many human diseases. To investigate a role for NF-κB in immunity and disease on a broader evolutionary scale we have characterized NF-κB in a sea anemone (Exaiptasia pallida; called Aiptasia herein) model for cnidarian symbiosis and dysbiosis (i.e., "bleaching"). We show that the DNA-binding site specificity of Aiptasia NF-κB is similar to NF-κB proteins from a broad expanse of organisms. Analyses of NF-κB and IκB kinase proteins from Aiptasia suggest that non-canonical NF-κB processing is an evolutionarily ancient pathway, which can be reconstituted in human cells. In Aiptasia, NF-κB protein levels, DNA-binding activity, and tissue expression increase when loss of the algal symbiont Symbiodinium is induced by heat or chemical treatment. Kinetic analysis of NF-κB levels following loss of symbiosis show that NF-κB levels increase only after Symbiodinium is cleared. Moreover, introduction of Symbiodinium into naïve Aiptasia larvae results in a decrease in NF-κB expression. Our results suggest that Symbiodinium suppresses NF-κB in order to enable establishment of symbiosis in Aiptasia. These results are the first to demonstrate a link between changes in the conserved immune regulatory protein NF-κB and cnidarian symbiotic status.

Structure-based prediction of C2H2 zinc-finger binding specificity: sensitivity to docking geometry.

Predicting the binding specificity of transcription factors is a critical step in the characterization and computational identification and of cis-regulatory elements in genomic sequences. Here we use protein-DNA structures to predict binding specificity and consider the possibility of predicting position weight matrices (PWM) for an entire protein family based on the structures of just a few family members. A particular focus is the sensitivity of prediction accuracy to the docking geometry of the structure used. We investigate this issue with the goal of determining how similar two docking geometries must be for binding specificity predictions to be accurate. Docking similarity is quantified using our recently described interface alignment score (IAS). Using a molecular-mechanics force field, we predict high-affinity nucleotide sequences that bind to the second zinc-finger (ZF) domain from the Zif268 protein, using different C2H2 ZF domains as structural templates. We identify a strong relationship between IAS values and prediction accuracy, and define a range of IAS values for which accurate structure-based predictions of binding specificity is to be expected. The implication of our results for large-scale, structure-based prediction of PWMs is discussed.

Structural alignment of protein--DNA interfaces: insights into the determinants of binding specificity.

A new method is introduced to structurally align interfaces observed in protein--DNA complexes. The method is based on a procedure that describes the interfacial geometry in terms of the spatial relationships between individual amino acid--nucleotide pairs. An amino acid--amino acid similarity matrix, S, is defined that provides a quantitative measure of the geometric relationships of amino acids in different interfaces and the entire stretch of "local" DNA within some distance of each amino acid. S is used as a substitution matrix in a dynamic programming algorithm that aligns the interfacial amino acids of the two complexes. The quality of the alignment is determined by an interface alignment score, IAS, that provides a quantitative measure of the similarity in the docking geometry between two protein--DNA complexes. We have clustered a large set of protein--DNA complexes based on their IAS values. In general, proteins within a single family form identifiable clusters. Subgroup clustering is often observed within families offering a fine-grained description of docking geometries. Although proteins with similar folds tend to dock in similar ways, important differences are observed even for structural motifs that almost perfectly align. Relationships are observed between the interfaces formed in cognate and non-cognate complexes involving the same proteins indicating a strong driving force to maintain certain contacts, even if this requires a distortion of the DNA. There are cases where inter-family similarities are greater than intra-family similarities. Our method offers the possibility of comparing different protein--DNA interfaces in a detailed, objective and quantitative fashion. This offers the possibility of new approaches to the description of the determinants of molecular recognition and to the prediction of protein and DNA sequence combinations that are optimal for binding.