The mammalian 5-AMP-activated protein kinase (AMPK) is related to a growing family of protein kinases in yeast and plants that are regulated by nutritional stress. We find the most prominent expressed form of the hepatic AMPK catalytic subunit (␣ 1 ) is distinct from the previously cloned kinase subunit (␣ 2 ). The ␣ 1 (548 residues) and ␣ 2 (552 residues) isoforms have 90% amino acid sequence identity within the catalytic core but only 61% identity elsewhere. The tissue distribution of the AMPK activity most closely parallels the low abundance 6-kilobase ␣ 1 mRNA distribution and the ␣ 1 immunoreactivity rather than ␣ 2 , with substantial amounts in kidney, liver, lung, heart, and brain. Both ␣ 1 and ␣ 2 isoforms are stimulated by AMP and contain noncatalytic ␤ and ␥ subunits. The liver ␣ 1 isoform accounts for approximately 94% of the enzyme activity measured using the SAMS peptide substrate. The tissue distribution of the ␣ 2 immunoreactivity parallels the ␣ 2 8.5-kilobase mRNA and is most prominent in skeletal muscle, heart, and liver. Isoforms of the ␤ and ␥ subunits present in the human genome sequence reveal that the AMPK consists of a family of isoenzymes.The 5Ј-AMP-activated protein kinase (AMPK) 1 was initially identified as a protein kinase regulating hydroxymethylglutaryl-CoA reductase (1). Subsequently, the AMPK was shown to phosphorylate hepatic acetyl-CoA carboxylase (2) and adipose hormone-sensitive lipase (3). The AMPK appears to act as a metabolic stress-sensing protein kinase switching off biosynthetic pathways when cellular ATP levels are depleted and when 5Ј-AMP rises in response to fuel limitation and/or hypoxia (4). Partial amino acid sequencing of hepatic AMPK (5, 6) revealed that it consists of 3 subunits, the catalytic subunit ␣ (63 kDa), and two noncatalytic subunits, ␤ (40 kDa) and ␥ (38 kDa).The AMPK is a member of the yeast SNF1 protein kinase subfamily that includes protein kinases present in plants, nematodes, and humans (5-9). The AMPK catalytic subunit, ␣, has strong sequence identity to the catalytic domain of the yeast protein kinase SNF1, which is involved in the induction of invertase (SUC2) under conditions of nutritional stress due to glucose starvation (10). Both Snf1p and the AMPK require phosphorylation by an activating protein kinase for full activity (11). The sequence relationship between Snf1p and AMPK led to the finding that these enzymes share functional similarities, both phosphorylating and inactivating yeast acetyl-CoA carboxylase (5,11,12). Nevertheless, the AMPK does not complement SNF1 in yeast (11), indicating that their full range of functions are not identical. The noncatalytic ␤ and ␥ subunits of AMPK are also related to proteins that interact with Snf1p; the ␤ subunit is related to the SIP1/SIP2/GAL83 family of transcription regulators and the ␥ subunit to SNF4 (also called CAT3) (6, 13). EXPERIMENTAL PROCEDURESPeptide Sequencing-Peptides were derived from rat and porcine ␣ 1 subunit of the AMPK, by in situ proteolysis (5), and sequenced on either an Ap...

Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R-Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrL567 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvrL567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R-Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R-Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant-pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.avirulence protein ͉ resistance protein P lants resist disease through a variety of preformed and induced barriers to infection. Among these is a genetically determined pathogen recognition system controlled by host resistance genes (R genes). In this gene-for-gene resistance system, plant R genes confer resistance to pathogen strains carrying corresponding avirulence (Avr) genes (so-called because their presence prevents growth on resistant plants). The recognition event involving the products of the R and Avr genes triggers host defense responses, including a localized host cell death or hypersensitive response (HR) that limits the spread of the pathogen from the infection site. Most known plant resistance proteins (R proteins) contain nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains, with the latter implicated in pathogen recognition (1). In contrast, Avr proteins are diverse, and many have pathogenicity effector functions that play important roles in enhancing infection (2). The antagonistic relationship between R and Avr genes results in coevolutionary conflict as selection favors evolution of resistance in plants and virulence in their pathogens.The Arabidopsis RPM1, RPS2, and RPS5 NBS-LRR-type R proteins confer r...

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Importin-␣ is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-␣, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-␣ and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX 10 -12 KRRK) that can be used to identify novel nuclear proteins.Nucleocytoplasmic transport occurs through nuclear pore complexes, large proteinaceous structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (ϳ25 MDa). The best characterized nuclear targeting signals are the conventional nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). The NLSs fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters separated by a spacer. Despite the variability, the conventional basic NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews, see Refs. 2-4). Importin-␣ (Imp␣) contains the NLSbinding site, and importin-␤ (Imp␤) is responsible for the translocation of the importin-substrate complex through the nuclear pore complex. Once inside the nucleus, Ran-GTP binds to Imp␤ and causes the dissociation of the import complex. Imp␣ becomes autoinhibited, and both importin subunits return to the cytoplasm separately without the import cargo. The directionality of nuclear import is conferred by an asymmetric distribution of the GTP-and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins.Imp␣ consists of two structural and functional domains, a short basic N-terminal Imp␤-binding domain (5-7) and a large NLS-binding domain built of armadillo (Arm) repeats (8). The structural basis of monopartite and bipartite NLS recognition by Imp␣ has been studied crystallographically in yeast and mouse Imp␣ proteins (9 -11). The two basic clusters of the bipartite NLSs bind to two separate binding sites on Imp␣, involving Arm repeats 1-4 and 4 -8, respectivel...

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-␣/␤ heterodimer. Importin-␣ contains the NLS binding site, whereas importin-␤ mediates the translocation through the nuclear pore. We characterized the interactions involving importin-␣ during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-␣ is shown to exist in a monomeric autoinhibited state (as- Nucleocytoplasmic transport occurs through nuclear pore complexes, large supramolecular structures that penetrate the double lipid layer of the nuclear envelope. Most macromolecules require an active, signal-mediated transport process that enables the passage of particles up to 25 nm in diameter (ϳ25 MDa). The first and best characterized nuclear targeting signals are the classical nuclear localization sequences (NLSs) 1 that contain one or more clusters of basic amino acids (1). NLSs do not conform to a specific consensus sequence and fall into two distinct classes termed monopartite NLSs, containing a single cluster of basic amino acids, and bipartite NLSs, comprising two basic clusters.Despite the sequence variability, the classical NLSs are recognized by the same receptor protein termed importin or karyopherin, a heterodimer of ␣ and ␤ subunits (for recent reviews see Refs. 2-6). Importin-␣ contains the NLS-binding site, and importin-␤ is responsible for the translocation of the importincargo complex through the pore. Transfer through the pore is facilitated by the proteins Ran (Ras-related nuclear protein) and nuclear transport factor-2. Once inside the nucleus, importin-␤ binds to Ran-GTP to effect the dissociation of the import complex; the importin subunits return to the cytoplasm separately and without the cargo. The directionality of nuclear import is thought to be conferred by an asymmetric distribution of the GTP-and GDP-bound forms of Ran between the cytoplasm and the nucleus. This distribution is in turn controlled by various Ran-binding regulatory proteins.Importin-␣ consists of two structural and functional domains, a short basic N-terminal importin-␤-binding (IBB) domain (7-9), and a large NLS-binding domain comprising armadillo (Arm) repeats (10). The monopartite NLSs bind at a major site located between the first and fourth Arm repeats and additionally at a minor site spanning repeats 4 -8 (11-13). The bipartite NLSs span the two binding sites, with each site recognizing one of the basic clusters (12, 13). The linker sequence between the two basic clusters makes few contacts with the receptor, consistent with its tolerance to mutations. The affinity of the importin-targeting sequence interaction is a critical parameter in determining transport efficiency (3).The structure of mouse importin-␣ showed that in the absence of importin-␤ or NLS-containing proteins, a part of the IBB domain occupies the major NLS-binding site (14). Based on this observation, it was sugges...

The gene-for-gene mechanism of plant disease resistance involves direct or indirect recognition of pathogen avirulence (Avr) proteins by plant resistance (R) proteins. Flax rust (Melampsora lini) AvrL567 avirulence proteins and the corresponding flax (Linum usitatissimum) L5, L6, and L7 resistance proteins interact directly. We determined the three-dimensional structures of two members of the AvrL567 family, AvrL567-A and AvrL567-D, at 1.4-and 2.3-Å resolution, respectively. The structures of both proteins are very similar and reveal a b-sandwich fold with no close known structural homologs. The polymorphic residues in the AvrL567 family map to the surface of the protein, and polymorphisms in residues associated with recognition differences for the R proteins lead to significant changes in surface chemical properties. Analysis of single amino acid substitutions in AvrL567 proteins confirm the role of individual residues in conferring differences in recognition and suggest that the specificity results from the cumulative effects of multiple amino acid contacts. The structures also provide insights into possible pathogen-associated functions of AvrL567 proteins, with nucleic acid binding activity demonstrated in vitro. Our studies provide some of the first structural information on avirulence proteins that bind directly to the corresponding resistance proteins, allowing an examination of the molecular basis of the interaction with the resistance proteins as a step toward designing new resistance specificities.

The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Impalpha. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Impalpha. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type I (HTLV-I) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal a-helix to the predicted N-terminal a-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal a-helical sequences.Keywords: E. coli protein expression; maltose-binding protein/HTLV-1 gp2 1 chimera; protein crystallization; trimerization; X-ray diffraction Crystallization and phase determination remain the two ratelimiting steps in determining protein crystal structures. The strategy of using fusion proteins comprising a "crystallization tag" of known three-dimensional structure and the protein of interest offers an approach to overcoming these problems. The crystallization conditions and crystal contacts found important in crystallization of the tag may be used to guide the crystallization of the fusion protein. Knowledge of the three-dimensional structure of the crystallization tag should then allow determination of the starting phases by the method of molecular replacement. Moreover, knowledge of the heavy atom-binding sites in the crystallization tag can be used to determine the starting phases by the methods of multiple isomorphous replacement and multi-wavelength anomalous dispersion. The strategy offers similar advantages as co-crystallization with antibody fragments (Air et al., 1987;Prongay et al., 1990;Jacobo-Molina et al., 1991;Ostermeier et al., 1995) while being