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Despite having irregular structure, protein loops often adopt specific conformations that are critical to protein function. Most studies in de novo protein design have focused on creating proteins with regular elements of secondary structure connected by very short loops or turns. To design longer protein loops that adopt specific conformations, we have developed a protocol within the Rosetta molecular modeling program that iterates between optimizing the sequence and conformation of a loop in search of low-energy sequence-structure pairs. We have tested the procedure by designing 10-residue loops for the connection between the second and third strand in the [beta]-sandwich protein tenascin. Three low-energy designs from 7,200 flexible backbone trajectories were selected for experimental characterization. All three designs, called LoopA, LoopB, and LoopC, adopt stable folded structures. High-resolution crystal structures of LoopA and LoopB have been solved. LoopB adopts a structure very similar to the design model (0.46 A rmsd), and all but one of the side chains are modeled in the correct rotamers. LoopA crystallized at low pH in a structure that differs dramatically from our design model. It forms a strand-swapped dimer mediated by hydrogen bonds to protonated glutamic acids. Gel filtration indicates that the protein is not a dimer at neutral pH. These results suggest that the high-resolution design of protein loops is possible; however, they also highlight how small changes in protein energetics can dramatically perturb the low free energy structure of a protein. computational protein design | loop modeling | tenascin

Phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. It remains unknown how individual PDE families selectively recognize cAMP and cGMP. This work reports structural studies on substrate specificity. The crystal structures of the catalytic domains of the D674A and D564N mutants of PDE10A2 in complex with cAMP and cGMP reveal that two substrates bind to the active site with the same syn configuration but different orientations and interactions. The products AMP and GMP bind PDE10A2 with the anti configuration and interact with both divalent metals, in contrast to no direct contact of the substrates. The structures suggest that the syn configurations of cAMP and cGMP are the genuine substrates for PDE10 and the specificity is achieved through the different interactions and conformations of the substrates. The PDE10A2 structures also show that the conformation of the invariant glutamine is locked by two hydrogen bonds and is unlikely to switch for substrate recognition. Sequence alignment shows a potential pocket, in which variation of amino acids across PDE families defines the size and shape of the pocket and thus determines the substrate specificity. crystal structure | cyclic nucleotides cAMP and cGMP

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. The crystal structure of the catalytic domain of PDF9A2, a member of a PDE family specifically hydrolyzing cGMP, has been determined at 2.23-[Angstrom] resolution. The PDE9A2 catalytic domain closely resembles the cAMP-specific PDE4D2 but is significantly different from the cGMP-specific PDE5A1, implying that each individual PDE family has its own characteristic substrate recognition mechanism. The different conformations of the H and M loops between PDE9A2 and PDE5A1 imply their less critical roles in nucleotide recognition. The nonselective inhibitor 3-isobutyl-1methylxanthine (IBMX) binds to a similar subpocket in the active sites of PDE4, PDE5, and PDE9 and has a common pattern of the binding. However, significantly different orientations and interactions of IBMXs are observed among the three PDE families and also between two monomers of the PDE9A2 dimer. The kinetic properties of the PDE9A2 catalytic domain similar to those of full-length PDE9A imply that the N-terminal regulatory domain does not significantly alter the catalytic activity and the IBMX inhibition.

Calcineurin, a [Ca.sup.2+]/calmodulin-dependent protein phosphatase, is the common target for two immunophilin--immunosuppressant complexes, cyclophilin A--cyclosporin A (CyPA-CsA) and FKBP--FK506. How the two structurally distinct immunophilin--drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-[Angstrom] resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike FKBP-FK506, CyPA-CsA interacts with Arg-122 at the active site of CN, implying direct involvement of CyPA-CsA in the regulation of CN catalysis. The simultaneous interaction of CyPA with both the composite surface and the active site of CN suggests that the composite surface may serve as a substrate recognition site responsible for the narrow substrate specificity of CN. The comparison of CyPA-CsA-CN with FKBP-FK506-CN significantly contributes to understanding the molecular basis of regulation of CN activity by the immunophilin--immunosuppressant. immunosuppressants | FK506 | FK506-binding protein | protein phosphatase | T cell

A comparison of the dipeptide and tetrapeptide complexes with cyclophilin A reveals differences in interactions at the active site. The binding of the dipeptides is similar to succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with respect to N-terminal hydrogen bonding and hydrophobic interactions. The hydrogen bonding between the carboxyl terminus of the peptides and Arg55, the side chain conformation of Arg55, and the binding of water at the active site are, however, different. The differences suggest that the dipeptides are probably inhibitors of peptidyl-prolyl cis-trans isomerases.

Cyclophilin A catalyzes the cis to trans isomerization of a peptidyl-prolyl amide bond. Crystal structure studies of human recombinant cyclophilin A complexed to a succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate indicate that only the cis form attachs to cyclophilin A. The trans form is unable to fit into the active site. The structure reveals that Arg55 hydrogen-bonds to the nitrogen and deconjugates the resonance of prolyl amide bond and helps in the cis-trans transition.

The crystal structure ofthe complex formed between fructose-2,6-bisphosphate and fructose 1,6- bisphosphatase (Fru-1,6-Pase) was determined at a resolution of 2.6 angstroms. The analysis showed that fructose-2,6-bisphosphate binds to the active sites ofthe C1 and C4 chains, the C2 and C3 chains, and to the AMP sites of the C1 and C4 chains, but not on the C2 and C3 chains of Fru-1,6-Pase. The model may be applied to the complex between Fru-1,6-Pase and fructose-1,6-bisphosphate, and allows for the presence of a water molecule bridging the 1-phosphate and the metal.

Results show that simultaneous interaction of two divalent metal ions with the phosphate group of AMP suggests a binuclear catalysis by phosphodiesterase as deduced from the crystal structure of the human cyclic nucleotide phosphodiesterase catalytic domain in complex with AMP at 2.4 Angstrom resolution. Further, structural data reveal that bridging two metal ions by a hydroxide ion or a water may form a nucleophile that hydrolyzes the cAMP phosphodiester bond.

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family., Cyclic nucleotides are intracellular second messengers that play a key role in many physiological processes. The levels of these nucleotides are tightly regulated at the point of synthesis by receptor-linked [...]