Toc34, a 34-kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex, which associates with precursor proteins during protein transport across the chloroplast outer membrane. Here we report the 2.0 A resolution crystal structure of the cytosolic part of pea Toc34 in complex with GDP and Mg2+. In the crystal, Toc34 molecules exist as dimers with features resembling those found in a small GTPase in complex with a GTPase activating protein (GAP). However, gel filtration experiments revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer solution at pH 7.2. Mutation of Arg 128, an essential residue for dimerization, to an Ala residue led to the formation of an exclusively monomeric species whose GTPase activity is significantly reduced compared to that of wild type Toc34. These results, together with a number of structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer.

Phosphoglucose isomerase (PGI) plays a central role in both the glycolysis and the gluconeogenesis pathways. We present here the complete crystal structure of PGI from Bacillus stearothermophilus at 2.3-Å resolution. We show that PGI has cell-motility-stimulating activity on mouse colon cancer cells similar to that of endogenous autocrine motility factor (AMF). PGI can also enhance neurite outgrowth on neuronal progenitor cells similar to that observed for neuroleukin. The results confirm that PGI is neuroleukin and AMF. PGI has an open twisted ␣͞␤ structural motif consisting of two globular domains and two protruding parts. Based on this substrate-free structure, together with the previously published biological, biochemical, and modeling results, we postulate a possible substrate-binding site that is located within the domains' interface for PGI and AMF. In addition, the structure provides evidence suggesting that the top part of the large domain together with one of the protruding loops might participate in inducing the neurotrophic activity.

Background: UppP, an integral membrane protein involved in the bacterial cell wall synthesis, catalyzes the dephosphorylation of undecaprenyl pyrophosphate. Results: The enzyme active site is proposed by modeling, molecular dynamics, and mutagenesis. Conclusion: The enzyme active-site, composed of (E/Q)XXXE and PGXSRSXXT motifs and a histidine, is proposed to be in the periplasm. Significance: This study provides a first insight into structure-function relationships of E. coli UppP.

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There have been ongoing efforts to develop more sensitive and fast quantitative screening of cancer markers by use of fluorometric immunochromatographic test strips (ICTS) since the remarkable advances in fluorescent nanomaterials. Semiconducting polymer dots (Pdots) have recently emerged as a new type of biocompatible fluorescent probe with extraordinary brightness which is suitable for biological and clinical use. Here, we developed Pdot-based ICTS for quantitative rapid screening of prostate-specific antigen (PSA), α-fetoprotein (AFP), and carcinoembryonic antigen (CEA) in 10 min. Through use of the ultrahigh fluorescence brightness of Pdots, this immunosensor enabled much better detection sensitivity (2.05, 3.30, and 4.92 pg/mL for PSA, AFP, and CEA, respectively), in which the detection limit is at least 2 orders of magnitude lower than that of conventional fluorometric ICTS. Furthermore, we performed proof-of-concept experiments for simultaneous determination of multiple tumor markers in a single test strip. These results demonstrated that this Pdot-based ICTS platform is a promising candidate for developing new generations of point-of-care diagnostics. To the best of our knowledge, this is the first example of Pdot-based ICTS with multiplexing capability.

Ym1, a secretory protein synthesized by activated murine peritoneal macrophages, is a novel mammalian lectin with a binding specificity to GlcN. Lectins are responsible for carbohydrate recognition and for mediating cell-cell and cell-extracellular matrix interactions in microbes, plants, and animals. Glycosaminoglycan heparin/heparan sulfate binding ability was also detected in Ym1. We report here the three-dimensional structure of Ym1 at 2.5-Å resolution by x-ray crystallography. The crystal structure of Ym1 consists of two globular domains, a ␤/␣ triose-phosphate isomerase barrel domain and a small ␣ ؉ ␤ folding domain. A notable electron density of sugar is detected in the Ym1 crystal structure. The saccharide is located inside the triosephosphate isomerase domain at the COOH terminal end of the ␤-strands. Both hydrophilic and hydrophobic interactions are noted in the sugar-binding site in Ym1. Despite the fact that Ym1 is not a chitinase, structurally, Ym1 shares significant homology with chitinase A of Serratia marcescens. Ym1 and chitinase A have a similar carbohydrate binding cleft. This study provides new structure information, which will lead to better understanding of the biological significance of Ym1 and its putative gene members.

The 70-kDa heat shock proteins (Hsp70), including the cognates (Hsc70), are molecular chaperones that prevent misfolding and aggregation of polypeptides in cells under both normal and stressed conditions. They are composed of two major structural domains: an N-terminal 44-kDa ATPase domain and a C-terminal 30-kDa substrate binding domain. The 30-kDa domain can be divided into an 18-kDa subdomain and a 10-kDa subdomain. Here we report the crystal structure of the 10-kDa subdomain of rat Hsc70 at 3.45 Å. Its helical region adopted a helix-loop-helix fold. This conformation is different from the equivalent subdomain of DnaK, the bacterial homologue of Hsc70. Moreover, in the crystalline state, the 10-kDa subdomain formed dimers. The results of gel filtration chromatography further supported the view that this subdomain was selfassociated. Upon gel filtration, Hsc70 was found to exist as a mixture of monomers, dimers, and oligomers, but the 60-kDa fragment was predominantly found to exist as monomers. These findings suggest that the ␣-helical region of the 10-kDa subdomain dictates the chaperone self-association.

Phosphoglucose isomerase catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. In addition, phosphoglucose isomerase has been shown to have functions equivalent to neuroleukin, autocrine motility factor, and maturation factor. Here we present the crystal structures of phosphoglucose isomerase complexed with 5-phospho-D-arabinonate and N-bromoacetylethanolamine phosphate at 2.5-and 2.3-Å resolution, respectively. The inhibitors bind to a region within the domains' interface and interact with a histidine residue (His 306 ) from the other subunit. We also demonstrated that the inhibitors not only affect the enzymatic activity of phosphoglucose isomerase, but can also inhibit the autocrine motility factor-induced cell motility of CT-26 mouse colon tumor cells. These results indicate that the substrate and the receptor binding sites of phosphoglucose isomerase and autocrine motility factor are located within close proximity to each other. Based on these two complex structures, together with biological and biochemical results, we propose a possible isomerization mechanism for phosphoglucose isomerase.Phosphoglucose isomerase (PGI) 1 (EC 5.3.1.9), a glycolytic enzyme, is an essential enzyme in all tissues. It interconverts glucose 6-phosphate and fructose 6-phosphate, hence plays a central role in both the glycolysis and the gluconeogenesis pathways. PGI deficiency in humans is an autosomal recessive genetic disorder that has the typical manifestation of nonspherocytic hemolytic anemia of variable clinical expression (1, 2). The serum activity of human PGI serves as a tumor marker in cancer patients (3, 4) and elevation in PGI activity is closely correlated with metastasis (5, 6). In addition to its essential role in carbohydrate metabolism in the cytoplasm, PGI also functions as neuroleukin (NLK) (7-9), autocrine motility factor (AMF) (10, 11), and maturation factor (MF) (12).NLK is a neurotrophic growth factor that promotes the survival of spinal and sensory neurons (13). Interestingly, PGI and NLK have previously been reported to have differential induction capabilities for certain neurons (9). AMF, a new class of cytokines, can stimulate cell migration in vitro and metastasis in vivo (10, 14 -16). Amino acid sequencing and immunological cross-reactivity experiments suggest that mouse AMF is identical or closely related to PGI/NLK (10). MF is capable of mediating the differentiation of human myeloid leukemic HL-60 cells to terminal monocytic cells, and apparently a high degree of homology exists between the MF of myeloid leukemia cells and PGI or NLK (12).It was previously suggested that PGI may recognize a sugarcontaining molecule(s) at the cell surface (7) and that NLK binds to the cell surface in a carbohydrate-dependent manner utilizing a PGI-like structure. Watanabe (10, 16) also pointed out that AMF (PGI/NLK/MF) may contain a PGI-like structure and initiates signal transduction by interacting with the carbohydrate side chains of the extracellular domain of the AMF receptor (AMF...