The ankyrin repeat as molecular architecture for protein recognition

The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.     

Structural and Functional Analysis of the GerD Spore Germination Protein of Bacillus Species

Spore germination in Bacillus species represents an excellent model system with which to study the molecular mechanisms underlying the nutritional control of growth and development. Binding of specific chemical nutrients to their cognate receptors located in the spore inner membrane triggers the germination process that leads to a resumption of metabolism in spore outgrowth. Recent studies suggest that the inner membrane GerD lipoprotein plays a critical role in the receptor-mediated activation of downstream germination events. The 121-residue core polypeptide of GerD (GerD^60-180) from Geobacillus stearothermophilus forms a stable α-helical trimer in aqueous solution. The 2.3-Å-resolution crystal structure of the trimer reveals a neatly twisted superhelical rope, with unusual supercoiling induced by parallel triple-helix interactions. The overall geometry comprises three interleaved hydrophobic screws of interacting helices linked by short turns that have not been seen before. Using complementation analysis in a series of Bacillus subtilis gerD mutants, we demonstrated that alterations in the GerD trimer structure have profound effects on nutrient germination. This important structure–function relationship of trimeric GerD is supported by our identification of a dominant negative gerD mutation in B. subtilis. These results and those of others lead us to propose that GerD mediates clustering of germination proteins in the inner membrane of dormant spores and thus promotes the rapid and cooperative germination response to nutrients.     

Design and characterization of a hyperstable p16INK4a that restores Cdk4 binding activity when combined with oncogenic mutations

Cyclin-dependent kinase inhibitor p16(INK4a) is the founding member of the INK4 family of tumor suppressors capable of arresting mammalian cell division. Missense mutations in the p16(INK4a) gene (INK4a/CDKN2A/MTS1) are strongly linked to several types of human cancer. These mutations are evenly distributed throughout this small, ankyrin repeat protein and the majority of them disrupt the native secondary and/or tertiary structure, leading to protein unfolding, aggregation and loss of function. We report here the use of multiple stabilizing substitutions to increase the stability of p16(INK4a) and furthermore, to restore Cdk4 binding activity of several defective, cancer-related mutant proteins. Stabilizing substitutions were predicted using four different techniques. The three most effective substitutions were combined to create a hyperstable p16(INK4a) variant that is 1.4 kcal/mol more stable than wild-type. This engineered construct is monomeric in solution with wild-type-like secondary and tertiary structure and cyclin-dependent kinase 4 binding activity. Interestingly, these hyperstable substitutions, when combined with oncogenic mutations R24P, P81L or V126D, can significantly restore Cdk4 binding activity, despite the divergent features of each destabilizing mutation. Extensive biophysical studies indicate that the hyperstable substitutions enhance the binding activity of mutant p16 through several different mechanisms, including an increased amount of secondary structure and thermostability, reduction in exposed hydrophobic surface(s) and/or a reduced tendency to aggregate. This apparent global suppressor effect suggests that increasing the thermodynamic stability of p16 can be used as a general strategy to restore the biological activity to defective mutants of this important tumor suppressor protein.     

Correction to: Exploratory Prognostic Biomarkers of Complement-Mediated Thrombotic Microangiopathy (CM-TMA) in Adults with Atypical Hemolytic Uremic Syndrome (aHUS): Analysis of a Phase III Study of Ravulizumab

Molecular Diagnosis & Therapy -