Titus J. Boggon

Regulated expression of Kruppel like factor (KLF) transcription factors is essential for normal maintenance of endothelial cells, but loss of either K-Rev interaction trapped 1 (KRIT1) or cerebral cavernous malformations 2 (CCM2) proteins results in significant over-expression of KLF4 protein, causing the cerebrovascular disorder, cerebral cavernous malformations. Here, combining knockdown and reconstitution in an endothelial cell line, with co-immunoprecipitation, biophysical analysis of purified proteins, and co-crystallography, we find that to restrain KLF4 expression, two CCM2 proteins must cluster on a single KRIT1, with the PTB domain of each CCM2 protein binding either the second or third NPxF motif within KRIT1. This clustering of two PTB domains to a single peptide reveals a previously unobserved mechanism for PTB domain recruitment to partner proteins. Overall, our data support a model where clustering of two CCM2 molecules to one KRIT1 is required for normal regulation of expression of KLF4 transcription factor.

Signaling downstream of Mitogen-Activated Protein Kinase Kinase Kinases (MAP3K) is promiscuous. In the vascular and immune systems the MAP3K, MEKK2, activates different substrates, but the mechanisms of substrate targeting have not been delineated. Here, we determine the 2.4 Å crystal structure of the MEKK2 kinase domain in complex with the small molecule inhibitor, ponatinib. We find that MEKK2 dimerizes by a surface centered on the αG helix and the C-terminal region of the activation segment, that this surface is important for MEKK2 autophosphorylation and dimerization, and that this surface is conserved with MEKK3. We then assess the importance of the surface for phosphorylation and recruitment of two MAP2K substrates, MEK5 and MKK6. We find that both MEK5 and MKK6 require the αG helix-mediated interaction for phosphorylation. In contrast, MEKK2 recruitment of MEK5 is dependent on PB1 domain interactions but MKK6 recruitment is associated with the αG helix-mediated interaction. Our study therefore provides a framework to understand diverse substrate targeting by the MAP3Ks, MEKK2 and MEKK3.

The RASA1 gene is mutated in cerebrovascular disorders and cancer, yet how the resulting mutations in the GTPase Activating Protein, RasGAP (p120RasGAP, RASA1) dysregulate signaling remains poorly understood. Here, we catalogue currently reported disease-associated mutations in RASA1 and assess their impact on RasGAP protein in vitro . On mapping these mutations onto experimental structures and structural models of RasGAP we identify regions that suggest functional impact. We assess key mutations within these regions for their effects on protein expression, thermal stability, and their interactions with a known binding partner, p190RasGAP. We then assess Michaelis-Menten kinetics of the mutant RasGAP proteins towards Ras. Together, we find that disease-associated RasGAP mutations classify into a panel of distinct classes based on their mode of dysregulation. We demonstrate that protein stability is necessary but not sufficient for full catalytic activity and that destabilizing mutations across the length of the protein can disrupt this function, but that the C2 domain appears to be unique in its role of regulating GAP activity by mechanisms other than destabilization involving the interactions of specific residues.

Introduction: Numerous X-ray crystal structures of the c-Src SH3 domain have provided a large sampling of atomic-level information for this important signaling domain. Multiple crystal forms have been reported, with variable crystal lattice contacts and chemical crystallization conditions. Materials and Methods: We crystallized the c-Src SH3 domain in a crystallization buffer containing NiCl2. Results: A unique crystal structure of the Src SH3 domain in the trigonal space group H32 is determined to 1.45 Å resolution. Crystal packing and anomalous scattering reveal that this crystal form is mediated by two ordered nickel ions provided by the crystallization buffer. Nickel coordination occurs in a 2:2 stoichiometry, which dimerizes two SH3 domain monomers across a pseudo-twofold rotation axis and involves the native N-terminal c-Src SH3 amino acid sequence, a surface-exposed histidine residue, and ordered water molecules. Discussion: This study provides an example of metal-mediated crystallization and metal binding by N-terminal protein residues, contrasting with the Amino-Terminal Copper and Nickel Binding (ATCUN) motif. Conclusion: Alternative avenues help widen the potential for future crystallography-based studies of the c-Src SH3 domain.

Ras and the small GTPase group are essential for myriad cellular processes and cycle between GDP- and GTP-loaded states to allow stringent control of downstream signaling pathways. Biochemical studies of small GTPases can therefore require a specific nucleotide-bound state. Small GTPases possess basal intrinsic activity to process GTP into GDP plus inorganic phosphate, therefore in vitro exchange of GDP for GTP is necessary for assays on GTP-loaded states. Here, we assess the methodology of in vitro nucleotide exchange for soluble H-Ras. We begin by describing a protocol to quantify the nucleotide bound content of H-Ras using anion exchange chromatography and use this protocol to investigate optimal strategies for loading Ras with GTP by assessing the effects of time, temperature, H-Ras concentration, magnesium, excess nucleotide, and isoform identity. We continue by considering storage of GTP-loaded H-Ras and present optimal conditions to minimize intrinsic GTP hydrolysis. Finally, we conclude by investigating the nucleotide composition of recombinantly expressed H-Ras encompassing cancer mutations at residues Gly12, Gly13, and Gln61. We therefore describe methodology to quantitatively analyze the nucleotide content of small GTPases and their mutants, and demonstrate conditions to achieve efficient GTP loading of Ras.

Numerous X-ray crystal structures of the c-Src SH3 domain have provided a large sampling of atomic-level information for this important signaling domain. Multiple crystal forms have been reported, with variable crystal lattice contacts and chemical crystallization conditions. Here, we report a unique crystal structure of Src SH3 domain in trigonal space group H 3 ₂ to 1.45 Å resolution. Crystal packing and anomalous scattering reveal that this crystal form is mediated by two ordered nickel ions provided by the crystallization buffer. Nickel coordination occurs in a 2:2 stoichiometry which dimerizes two SH3 domain monomers across a pseudo-twofold rotation axis and involves the native N-terminal c-Src SH3 amino acid sequence, a surface-exposed histidine residue, and ordered water molecules. This study provides an example of metal binding by N-terminal protein residues that contrasts the amino terminal copper and nickel binding (ATCUN) motif and is an alternative avenue for crystallization of the Src SH3 domain. STRUCTURED Introduction: Numerous X-ray crystal structures of the c-Src SH3 domain have provided a large sampling of atomic-level information for this important signaling domain. Multiple crystal forms have been reported, with variable crystal lattice contacts and chemical crystallization conditions. Materials and Methods: We crystallize the c-Src SH3 domain in a crystallization buffer containing NiCl ₂ . Results: A unique crystal structure of Src SH3 domain in trigonal space group H 3 ₂ to 1.45 Å resolution is determined. Crystal packing and anomalous scattering reveal that this crystal form is mediated by two ordered nickel ions provided by the crystallization buffer. Nickel coordination occurs in a 2:2 stoichiometry which dimerizes two SH3 domain monomers across a pseudo-twofold rotation axis and involves the native N-terminal c-Src SH3 amino acid sequence, a surface-exposed histidine residue, and ordered water molecules. Discussion: This study provides an example of metal-mediated crystallization and metal binding by N-terminal protein residues that contrasts the amino terminal copper and nickel binding (ATCUN) motif. Conclusion: Alternative avenues for helps widen the potential for future crystallography-based studies of the c-Src SH3 domain.