p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K d 0.6 M) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K d 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.GTP-binding proteins of the Ras superfamily are molecular switches which cycle between the GDP-bound off and GTPbound on states. In the on state, they interact with effectors which are defined as molecules interacting more tightly with the GTP-bound form than with the GDP-bound form. By interacting with effectors, they mediate downstream biological effects. The switch returns to the GDP-bound off state by the GTPase reaction which is catalyzed by GTPase-activating proteins (4, 5).Recently, it has become clear that many of the GTP-binding proteins interact with an array of different effectors and thus are involved in more than one signal transduction pathway (26). Many of these effectors are protein kinases that become activated in the course of this interaction. A prominent example are the different Raf kinases (three isoforms) which interact with activated Ras in the course of growth regulation and become activated via a mechanism that involves translocation to the plasma membrane and most likely further allosteric regulatory events which are still incompletely understood.In the case of the Rho subfamily members Rho, Rac, and Cdc42, a number of Ser/Thr-specific protein kinases have been identified, such as protein kinase N and the Rho kinases for Rho. The first kinases to be identified as downstream targets were the Tyr-specific ACK (activated Cdc42-associated kinase) (23) and the Ser/Thr-specific PAK (p21-activated kinase) (24). PAK constitutes a large family of related protein k...

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix.

Most glucoamylases (α‐1,4‐d‐glucan glucohydrolase, EC 3.2.1.3) have structures consisting of both a catalytic and a starch binding domain. The structure of a glucoamylase from Saccharomycopsis fibuligera HUT 7212 (Glu), determined a few years ago, consists of a single catalytic domain. The structure of this enzyme with the resolution extended to 1.1 Å and that of the enzyme–acarbose complex at 1.6 Å resolution are presented here. The structure at atomic resolution, besides its high accuracy, shows clearly the influence of cryo‐cooling, which is manifested in shrinkage of the molecule and lowering the volume of the unit cell. In the structure of the complex, two acarbose molecules are bound, one at the active site and the second at a site remote from the active site, curved around Tyr464 which resembles the inhibitor molecule in the ‘sugar tongs’ surface binding site in the structure of barley α‐amylase isozyme 1 complexed with a thiomalto‐oligosaccharide. Based on the close similarity in sequence of glucoamylase Glu, which does not degrade raw starch, to that of glucoamylase (Glm) from S. fibuligera IFO 0111, a raw starch‐degrading enzyme, it is reasonable to expect the presence of the remote starch binding site at structurally equivalent positions in both enzymes. We propose the role of this site is to fix the enzyme onto the surface of a starch granule while the active site degrades the polysaccharide. This hypothesis is verified here by the preparation of mutants of glucoamylases Glu and Glm.