Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Fc receptors, which are expressed on the majority of hematopoietic cells, play important roles in antibody-mediated immune responses. [3][4][5]. In addition to variations in affinity, each receptor displays distinct IgG subtype specificities. Unlike the high affinity receptors that can bind monomeric antibodies, the low affinity receptors preferentially bind to and are activated by immune complexes.Human Fc␥RIII exists as two isoforms, Fc␥RIIIA and Fc␥RIIIB, that share 96% sequence identity in their extracellular immunoglobulin-binding regions. Fc␥RIIIA is expressed on macrophages, mast cells, and natural killer cells as a transmembrane receptor. In contrast, Fc␥RIIIB, present exclusively on neutrophils, is anchored by a glycosyl-phosphatidylinositol linker to the plasma membrane. Although Fc␥RIIIA associates with the immunoreceptor tyrosine-based activation motif containing Fc⑀RI ␥-chain or the T cell receptor -chain for its signaling, Fc␥RIIIB lacks a signaling component. Nevertheless, it plays an active role in triggering Ca 2ϩ mobilization and in neutrophil degranulation (6, 7). In addition, Fc␥RIIIB, in conjunction with Fc␥RIIA, activates phagocytosis, degranulation, and the oxidative burst that leads to the clearance of opsonized pathogens by neutrophils. A soluble form of Fc␥RIIIB was reported to activate the CR3 complement receptordependent inflammatory process (8).The Fc binding region on Fc␥RII and Fc␥RIII has been identified through the work of chimeric receptors with Fc⑀RI as primarily the membrane proximal domain, including both the BC and FG loops. Further site-directed mutations have revealed several residues of the receptor critical to Fc binding (9 -11). Similar regions on the ␣-chain of Fc⑀RI were also identified to be critical for IgE binding affinity (12). The receptor binding site on Fc has been located through the construction of chimeric IgG molecules and mutational analysis at the lower hinge region, residues located in the hinge region between the C H 1 and C H 2 domains and immediately adjacent to the N terminus of the C H 2 domain of IgG (13-15). In particular, residues 234 -238 (Leu-Leu-Gly-Gly-Pro) of the lower hinge of IgG1 have been implicated in the receptor binding. The corresponding region of IgE has also been implicated in the Fc⑀RI binding (16). Apart from the lower hinge region, a few residues on the C H 2 domain of an IgG2b were also suggested to interact * This work was supported by the intramural research funding of NIAID, National Institutes of Health and by INSERM, Institut Curie, France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The recent crystal structures of Fc⑀RI␣, Fc␥RIIA, and Fc␥RIIB have each revealed a conserved Ig-like structure, with particularly the small hinge angle between the two Ig-like domains, which is unique to the Fc receptors (19 -21). We report here the crystal stru...

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Kinetoplast DNA (kDNA), the unusual mitochondrial DNA of Trypanosoma brucei, is a network containing thousands of catenated circles. Database searching for a kDNA replicative polymerase (pol) revealed no mitochondrial pol gamma homolog. Instead, we identified four proteins (TbPOLIA, IB, IC, and ID) related to bacterial pol I. Remarkably, all four localized to the mitochondrion. TbPOLIB and TbPOLIC localized beside the kDNA where replication occurs, and their knockdown by RNA interference caused kDNA network shrinkage. Furthermore, silencing of TbPOLIC caused loss of both minicircles and maxicircles and accumulation of minicircle replication intermediates, consistent with a role in replication. While typical mitochondria contain one DNA polymerase, pol gamma, trypanosome mitochondria contain five such enzymes, including the previously characterized pol beta.

Kinetoplast DNA (kDNA) is the remarkable mitochondrial genome of trypanosomatids. Its major components are several thousands of topologically linked DNA minicircles, whose replication origins are bound by the universal minicircle sequence-binding protein (UMSBP). The cellular function of UMSBP has been studied in Trypanosoma brucei by using RNAi analysis. Silencing of the trypanosomal UMSBP genes resulted in remarkable effects on the trypanosome cell cycle. It significantly inhibited the initiation of minicircle replication, blocked nuclear DNA division, and impaired the segregation of the kDNA network and the flagellar basal body, resulting in growth arrest. These observations, revealing the function of UMSBP in kDNA replication initiation and segregation as well as in mitochondrial and nuclear division, imply a potential role for UMSBP in linking kDNA replication and segregation to the nuclear S-phase control during the trypanosome cell cycle.kDNA replication initiation ͉ kDNA segregation ͉ kinetoplast DNA ͉ trypanosomes cell cycle control ͉ universal minicircle sequence-binding protein K inetoplast DNA (kDNA) is a unique extrachromosomal DNA, found in the single mitochondrion of trypanosomatids. It consists, in the different species, of a few dozen maxicircles (20-40 kb each) and a few thousand minicircles (0.5-10 kb each), that are interlocked topologically into a DNA network (1, 2). Minicircles in most trypanosomatid species contain two short sequences that are associated with replication initiation: a dodecamer, designated the universal minicircle sequence (UMS), and a hexamer. These sequences have been mapped to the replication origins of the minicircle's light (L) and heavy (H) strands, respectively. kDNA replication occurs during S phase of the cell cycle, approximately in parallel with nuclear DNA replication (3). Minicircles are released from the network into the kinetoflagellar zone, located in the mitochondrial matrix, between the kDNA network and the flagellar basal body. Each minicircle replicates as an individual replicon, forming gapped and nicked progeny molecules. Minicircle replication intermediates then migrate onto two antipodal sites, flanking the kDNA disk, in which primer-removal, repair of the gaps between Okazaki fragments and reattachment of the progeny minicircles to the network occurs. The final gap-filling and sealing of the topologically linked minicircles take place before the network division (recently reviewed in refs. 1 and 2).We have previously reported the presence in Crithidia fasciculata of a UMS-binding protein (UMSBP). The protein, which contains five tandemly arranged CCHC-type zinc-finger motifs, has been purified from cell extracts, and its encoding gene and genomic locus were cloned and analyzed (4-7). Genes encoding orthologous proteins have been identified in other trypanosomatid species [ref. 8 and supporting information (SI) Table 1]. UMSBP binds specifically the two conserved sequences, located at the minicircle replication origins: the UMS dodecamer and an H-st...

ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA). RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

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Transforming growth factor beta (TGF-beta) is involved in a wide range of biological functions including development, carcinogenesis, and immune regulation. Here we report the 1.1 A resolution crystal structure of human TGF-beta type II receptor ectodomain (TBRII). The overall structure of TBRII is similar to that of activin type II receptor ectodomain (ActRII) and bone morphogenic protein receptor type IA (BRIA). It displays a three-finger toxin fold with fingers formed by the beta strand pairs beta1-beta2, beta3-beta4, and beta5-beta6. The first finger in the TBRII is significantly longer than in ActRII and BRIA and folds tightly between the second finger and the C terminus. Surface charge distributions and hydrophobic patches predict potential TBRII binding sites.