Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

No abstract

No abstract

Highlights d N-glycosylation transforms a chaperone, GRP94, from a folder into a scaffolding protein d These changes are pathologic in nature as they remodel proteome-wide connectivity d The N-glycosylated GRP94 variant is a small and distinct fraction of the GRP94 pool d Proteome dysfunctions mediated by the N-glycosylated GRP94 variant are actionable

Summary Hsp90 chaperones undergo ATP-driven conformational changes during the maturation of client proteins, populating a “closed” state upon ATP binding in which the N-terminal domains of the homodimer form a second inter-protomer dimer interface. A structure of GRP94, the endoplasmic reticulum hsp90, in a closed conformation has not been described, and the determinants that regulate closure are not well understood. Here, we determined the 2.6 Å structure of AMPPNP-bound GRP94 in the closed dimer conformation. The structure includes the pre-N domain, a region preceding the N-terminal domain that is highly conserved in GRP94, but not in other hsp90s. We show that the GRP94 pre-N domain is essential for client maturation, and identify the pre-N domain as an important regulator of ATPase rates and dimer closure. The structure also reveals a GRP94:polypeptide interaction that partially mimics a client-bound state. The results provide structural insight into the ATP-dependent client maturation process of GRP94.

Lipid A from the nitrogen-fixing bacterium Rhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1-and 4-phosphate groups but is derivatized with a galacturonic acid substituent at position 4. R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked ␤-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked ␤-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IV A , and (3-deoxy-D-manno-octulosonic acid) 2 -lipid IV A . The enzyme does not cleave E. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes of Pseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IV A and (3-deoxy-D-manno-octulosonic acid) 2 -lipid IV A .1 H NMR spectroscopy of the deacylase reaction product, generated with lipid IV A as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked ␤-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.

Desosamine (1) is a 3-amino-3,4,6-trideoxyhexose found in several macrolide antibiotics such as methymycin (2), neomethymycin (3), pikromycin, and erythromycin. 1 Since modifying the structure of the sugar component holds promise for varying and/ or enhancing the biological activities of the parent antimicrobial agents, 2 a detailed understanding of the biosynthesis of desosamine is essential for developing suitable strategies to control, mimic, or alter its formation. Toward these goals, we have recently cloned and sequenced the entire desosamine biosynthetic gene cluster from the methymycin/neomethymycin producing strain, Streptomyces Venezuelae. 3 As shown in Scheme 1, eight of the nine open reading frames (ORFs) in this region have been assigned to steps in the biosynthesis of TDP-D-desosamine (7), while the remaining ORF, desR, which encodes a macrolide -glucosidase, appears to be involved in a glycosylationdeglycosylation self-resistance mechanism. 3a These assignments are based on sequence similarities to other sugar biosynthetic genes, especially those derived from the erythromycin cluster that has been independently characterized by Gaisser et al., 4a SalahBey et al., 4b and Summers et al. 4c Of particular interest in this cluster is the presence of two genes, desI and desV, both of which display sequence homology to coenzyme B 6 -dependent catalysts. 5 The encoded proteins of these two genes have been assigned to catalyze two of the key reactions in this pathwaysDesV may be an aminotransferase responsible for the C-3 transamination, whereas DesI is likely a dehydrase involved in the C-4 deoxygenation. 3,4 Depending on the order of these two key steps, two possible pathways have been proposed for the biosynthesis of TDP-Ddesosamine (7). 3,4 As shown in Scheme 1 pathway A, the C-4 deoxygenation, postulated to be mediated by the 4-dehydrase (DesI) and a putative reductase (DesII), 6 takes place on 4 to give 5. This is followed by C-3 transamination catalyzed by DesV to afford 6. 3,4c Alternatively, the C-3 transamination catalyzed by DesV may occur first to generate 3-aminosugar intermediate 8, which is then processed by the 4-dehydrase (DesI) and the reductase (DesII) to give 6 (Scheme 1, pathway B). 4a,b The mechanisms proposed for C-4 deoxygenation in both pathways are essentially alike and are based on the well-established C-3 deoxygenation in the biosynthesis of 3,6-dideoxyhexoses. 7 However, the substrate specificity and the cofactor requirements for the 4-dehydrase are clearly different in each case. 8 Although isolation and characterization of these enzymes would provide direct evidence to distinguish these mechanistic possibilities, due to problems encountered in expressing the corresponding genes to give soluble proteins, an approach relying on gene deletion and phenotype correlation was adapted to determine the sequence of events in this pathway and to define the chemical nature of the substrate for the 4-dehydrase.First, a double crossover mutant of S. Venezuelae (KdesV-41) in which the d...

The lpxK gene has been proposed to encode the lipid A 4-kinase in Escherichia coli (Garrett, T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997) J. Biol. Chem. 272, 21855-21864). In cell extracts, the kinase phosphorylates the 4-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet been purified because of instability. lpxK is co-transcribed with an essential upstream gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters. msbA may be involved in the transport of newly made lipid A from the inner surface of the inner membrane to the outer membrane. Insertion of an ⍀-chloramphenicol cassette into msbA also halts transcription of lpxK. We have now constructed a strain in which only the lpxK gene is inactivated by inserting a kanamycin cassette into the chromosomal copy of lpxK. This mutation is complemented at 30°C by a hybrid plasmid with a temperature-sensitive origin of replication carrying lpxK ؉ . When this strain (designated TG1/ pTAG1) is grown at 44°C, the plasmid bearing the lpxK ؉ is lost, and the phenotype of an lpxK knock-out mutation is unmasked. The growth of TG1/pTAG1 was inhibited after several hours at 44°C, consistent with lpxK being an essential gene. Furthermore, 4-kinase activity in extracts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4-kinase substrate) accumulated in TG1/pTAG1 cells grown at 44°C. The DS-1-P from TG1/ pTAG1 was isolated, and its structure was verified by 1 H NMR spectroscopy. DS-1-P had not been isolated previously from bacterial cells. Its accumulation in TG1/ pTAG1 provides additional support for the pathway of lipid A biosynthesis in E. coli. Homologs of lpxK are present in the genomes of other Gram-negative bacteria.