No abstract

An amino-terminal fragment of human apolipoprotein E3~residues 1-165! has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic~P2 1 2 1 2 1 ! crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E~residues 1-191!. A second orthorhombic crystal form~P2 1 2 1 2 1 with cell dimensions differing from the first form! and a trigonal~P3 1 21! crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing ␥ 1 , (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (< ϳ1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS. At higher AMPK concentrations, SAXS revealed concentration-dependent, reversible dimerization of AMPK heterotrimers and formation of higher oligomers, also confirmed by STEM mass measurements. Single particle reconstruction and averaging by SAXS and TEM, respectively, revealed similar elongated, flat AMPK particles with protrusions and an indentation. In the lower AMPK concentration range, addition of AMP resulted in a significant decrease of the radius of gyration by ϳ5% in SAXS, which indicates a conformational switch in AMPK induced by ligand binding. We propose a structural model involving a ligand-induced relative movement of the kinase domain resulting in a more compact heterotrimer and a conformational change in the kinase domain that protects AMPK from dephosphorylation of Thr 172 , thus positively affecting AMPK activity.Mammalian AMP-activated protein kinase (AMPK) 4 and its orthologs found in yeast, plants, insects, invertebrates, and vertebrates are fuel sensors of the eukaryotic cell and function as master regulators of energy metabolism (1-5). AMPK is a serine/threonine protein kinase, consisting of one catalytic ␣ and two regulatory subunits (␤ and ␥), that exist as multiple isoforms (␣ 1 , ␣ 2 , ␤ 1 , ␤ 2 , ␥ 1 , ␥ 2 , ␥ 3 ) and splice variants (␥ 2 and ␥ 3 ), thus allowing for the generation of multiple heterotrimeric isoform combinations. Critical for activation of AMPK is its phosphorylation at Thr 172 in the kinase domain of the ␣-subunit by either of the two upstream kinases of AMPK, LKB1-MO25␣-STRAD␣ or Ca 2ϩ /calmodulin-dependent protein kinase kinase ␤ (CaMKK␤) (6 -10). In addition, AMP allosterically stimulates AMPK (10, 11) by binding to two pairs of CBS domains in the ␥-subunits, called Bateman domains (12). These domains were reported to show high affinity for AMP (10, 13) and lower affinity for ATP (12), although a recent study indicates that these affinities may be similar (14). AMP is a very sensitive signal for an altered cellular energy status (13,15,16). Its intracellular concentration changes by 1 order of magnitude upon a 1% change in the cellular ATP concentration, due to the equilibrium reactions of adenylate kinase and creatine kinase (5, 15-18). The latter uses phosphocreatine to rapidly rephosphorylate ADP to ATP and thus maintains a high ATP/ADP ratio (17,...