Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical β-propeller domain

Proteins of the echinoderm microtubule-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase microtubule network. They contain a unique hydrophobic EML protein (HELP) motif and a variable number of WD40 repeats. Recurrent gene rearrangements in nonsmall cell lung cancer fuse EML4 to anaplastic lymphoma kinase (ALK), causing expression of several fusion oncoprotein variants. We have determined a 2.6-Å crystal structure of the representative ∼70-kDa core of EML1, revealing an intimately associated pair of β-propellers, which we term a TAPE (tandem atypical propeller in EMLs) domain. One propeller is highly atypical, having a discontinuous subdomain unrelated to a WD40 motif in place of one of its blades. This unexpected feature shows how a propeller structure can be assembled from subdomains with distinct folds. The HELP motif is not an independent domain but forms part of the hydrophobic core that joins the two β-propellers. The TAPE domain binds α/β-tubulin via its conserved, concave surface, including part of the atypical blade. Mapping the characteristic breakpoints of each EML4-ALK variant onto our structure indicates that the EML4 TAPE domain is truncated in many variants in a manner likely to make the fusion protein structurally unstable. We found that the heat shock protein 90 (Hsp90) inhibitor ganetespib induced degradation of these variants whereas others lacking a partial TAPE domain were resistant in both overexpression models and patient-derived cell lines. The Hsp90-sensitive EML4-ALK variants are exceptions to the rule that oncogenic fusion proteins involve breakpoints in disordered regions of both partners.Rearrangements in the short arm of chromosome 2 leading to genetic fusions between EML4 [echinoderm microtubule-associated protein (EMAP)-like 4] and the gene encoding anaplastic lymphoma kinase (ALK) occur in ∼5% of cases of nonsmall cell lung cancer (NSCLC) (1). Multiple variants of the EML4-ALK fusion have been identified in NSCLC resulting from translocations at different points within the EML4 gene (2). A similar genetic fusion has also been reported between EML1 and the gene encoding another tyrosine kinase, Abelson 1 (ABL1), in T-cell acute lymphoblastic leukemia (3). These fusions display potent transforming activity due to constitutive activation of the tyrosine kinase but confer addiction to the oncogene, and inhibition of the tyrosine kinase induces apoptosis in transformed cells (4, 5). The ALK inhibitor crizotinib is a highly effective first-line therapy for NSCLC patients with EML4-ALK fusions, but because the acquirement of resistance is inevitable, often involving secondary mutations in the kinase portion of the fusion protein, additional therapeutic strategies must be identified (6, 7).Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are being investigated as potential cancer therapeutics because many oncoproteins are obligate Hsp90 clients and they are rapidly degraded by the proteasome when Hsp90 is inhibited (8). Hsp90 inhibitors induce the degradation of EML4-ALK variant 1 and regression in some EML4-ALK–positive tumor models (7, 9, 10). Furthermore, clinical efficacy of an Hsp90 inhibitor in EML4-ALK NSCLC has been confirmed (11), and clinical trials are ongoing. However, because neither ALK nor EML4 are native Hsp90 clients, it was proposed that Hsp90 sensitivity of EML4-ALK fusions was due to their protein-folding properties, which might expose hydrophobic residues that lead to Hsp90 recruitment (12). EML4-ALK variants incorporating various portions of EML4 differ in sensitivity to Hsp90 inhibitors, suggesting that they have different protein-folding properties (12).In contrast to the abundance of studies on EML4-ALK fusions, the basic functions of EML proteins are relatively understudied. The archetypal EML protein was identified as the most abundant MAP in dividing echinoderm eggs and embryos, where it promotes microtubule dynamics (13, 14). Microtubule interactions of EML1-4 have been characterized in six EML proteins present in humans (15–18). More members of the family have been identified in Drosophila and Caenorhabditis elegans, which each have a single homolog (19). The EML proteins contain a variable number of WD40 repeats, and in EML1-4 these fall within an ∼70-kDa core region that begins with a conserved ∼60-amino-acid sequence that has been termed the hydrophobic EML protein (HELP) motif (15). Many EML proteins also contain a predicted coiled-coil (CC) in their N termini that is likely to mediate oligomerization.Functional studies of EML proteins and their oncogenic fusions have had to be interpreted without knowledge of their molecular structures. For example, it has been assumed that the HELP motif forms an independent domain that perhaps mediates specific interactions with microtubules (MTs) or the plasma membrane (15, 17, 18, 20). The extent to which the folding of the WD40 repeat region is disrupted in EML4-ALK fusions is also unclear because the number, position, and arrangement of β-propellers in EML proteins are unknown. We determined the crystal structure of the ∼70-kDa core HELP/WD region of human EML1. The structure serves as an archetype for the whole family and has an unusual molecular architecture that we have termed the TAPE (tandem atypical propeller in EMLs) domain. The structure enabled us to dissect the molecular determinants of tubulin-binding and map the precise location of breakpoints in EML4-ALK patients onto the structure. We explain how the partial EML moieties that are present in each of the variants confer differences between them with respect to Hsp90 inhibitor sensitivity.     

Parkinson disease-associated mutation R1441H in LRRK2 prolongs the “active state” of its GTPase domain

Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (Roc_R1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD) (1–5). LRRK2 is a large (2,527-aa) multidomain protein consisting of seven putative domains (2), including a Ras-like GTPase domain called Ras of complex proteins (Roc), followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear how perturbations of these activities result in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild type; therefore, its overactivation might be associated with disease pathogenesis (6).The tandem Roc-COR-Kin arrangement suggests that their activities might be coupled such that the GTPase activity of Roc might modulate the kinase activity. Indeed, several studies have shown that GTP binding to the Roc domain regulates the activity of the Kin domain (7, 8). Moreover, a PD-associated mutation in the Roc domain (R1441C) has been shown to have higher kinase activity (9), thus suggesting that mutations in the Roc domain, also up-regulate kinase activity.Understanding the function of Roc and its mechanism of action is important for understanding the mechanism of PD pathogenesis and therapeutic development. However, because of the lack sufficient quantity of protein samples amendable for detailed investigations, the biochemical properties and enzymatic activities of the Roc domain of LRRK2 are poorly understood.Here, we describe a stably folded construct of human Roc domain that enabled us to investigate quantitatively its biochemical and enzymatic properties. The results revealed that a PD-causing mutation R1441H in the Roc domain renders it less active at hydrolyzing GTP, as well as having higher affinity for GTP, than its wild-type counterpart, thereby increasing the residence time of its GTP-bound “active state,” which is associated with PD pathogenesis (8).     

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components

Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as water-soluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a β-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting β2 and β3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal β-strands (amino-latch) of the adjacent protomer, releasing the transmembrane stem domain folded into a β-sheet in the monomer (prestem), and interaction with the adjacent protomer.Pathogenic bacteria secrete various virulence factors to attack host cells. The pore-forming toxins (PFTs) are among the most sophisticated virulence factors, and are expressed as water-soluble monomeric proteins that assemble on the membranes of the target cells to form bilayer-spanning pores (1). With the appearance of the pore on the membrane, the cells are killed through leakage. It is interesting to note that PFTs are expressed not only by bacteria but also by eukaryotes, such as the immune proteins perforin and complement C9, suggesting the universality of these molecules in a wide range of organisms (2). PFTs can be classified into two families according to the secondary structure of the transmembrane region in the pore structure; i.e., α-helical PFT (α-PFT) and β-barrel PFT (β-PFTs) (3, 4).Staphylococcus aureus, a ubiquitous and pernicious human pathogen, secretes several β-PFTs including αHL, γ-hemolysin (γHL), leukocidin (LUK), and Panton–Valentine leukocidin (PVL) (5). The αHL consists of a single polypeptide, whereas the others are bicomponent β-PFTs that require the synergistic association of a class F component and a class S component. The γHL, LUK, and PVL are composed of LukF and Hlg2, LukF and LukS, and LukF-PV and LukS-PV, as class F and S components, respectively. The components of bicomponent β-PFTs are similar to each other and to αHL in amino acid sequence: Within a class, S and F proteins are approximately 70% identical, whereas between classes the identity drops to approximately 30%. Class F proteins are more closely related to αHL (approximately 30%) than class S proteins (approximately 20%) (5, 6). Extensive experiments have been carried out for more than two decades, and have suggested that the pore formation mechanism of bicomponent toxins is as follows (7, 8). The soluble forms of F and S components bind sequentially to the target cells and form a heterodimer (9, 10). Each heterodimer assembles into an oligomer on the target cell to form a ring-shaped particle called a prepore, in which the β-barrel pore is not yet formed (11–14). After forming a stable prepore, the β-barrel pore is formed. Pore formation requires the binding of phosphatidylcholine (PC) head groups to a cleft in the LukF component surrounded by Trp177 and Arg198 (Trp176 and Arg197 of LukF-PV) (13, 15, 16). The crystal structures of the monomeric forms of bicomponent β-PFTs [i.e., LukF (15), LukF-PV (17), and LukS-PV (18)], have been determined. However, the structures of the pore forms have not been reported at atomic resolution, which has hindered detailed discussion of the complicated molecular mechanism of action of bicomponent pore-forming toxins. Although bicomponent PFTs are found in several species, such as the edible mushroom Pleurotus ostreatus (19), the structures of these pores have not been reported.One of the most important issues for staphylococcal bicomponent PFTs is the stoichiometry of the class F and S components. Electron microscopy and cross-linking experiments of purified γHL pores on human erythrocyte membranes demonstrated the existence of a heptamer with a 3∶4 or 4∶3 molar ratio of F to S components (20, 21), whereas biochemical analyses of pores of engineered covalent γHL heterodimers on erythrocytes and leukocytes suggested octameric stoichiometry (22). Several reports using LUK pores formed on rabbit erythrocytes also demonstrated the existence of an octamer consisting of 4-plus-4 subunits (23–25). However, hexamer was also proposed based on structure modeling using monomeric structures of PVL (17) and electron microscopy of LUK pores on leukocytes and erythrocytes (26). It is also important to determine the significance of using two components. Although a role was proposed for the F component in initiating the pore formation process (27), that of the S component remains unclear.In the present study, we determined the crystal structure of the pore form of bicomponent β-PFT, γHL. This is a unique report of the crystal structure of a heterocomponent β-barrel- type transmembrane protein. This is also a unique bicomponent β-PFT of which both monomer- and pore-form structures have been determined by X-ray crystallography, which allowed us to discuss the pore formation mechanism based on their real structures at atomic resolution. Based on the structural differences between pore and monomer forms in combination with biological data accumulated over the past two decades, we propose a mechanism of pore formation by β-PFTs along with the roles of each component. The electrochemical properties of the pore are also discussed from a structural viewpoint.     

Autosomal recessive segregation of a truncating mutation of anti-Müllerian type II receptor in a family affected by the persistent Müllerian duct syndrome contrasts with its dominant negative activity in vitro

Anti-Müllerian hormone belongs to the TGFbeta family whose members exert their effects by signaling through two related serine/threonine kinase receptors. Mutations of the anti-Müllerian hormone type II receptor occur naturally, causing the persistent Müllerian duct syndrome. In a family with two members with persistent Müllerian duct syndrome and one normal sibling, we detected two novel mutations of the anti-Müllerian hormone type II receptor gene. One, transmitted by the mother to her three sons, is a deletion of a single base leading to a stop codon, causing receptor truncation after the transmembrane domain. The other, a missense mutation in the substrate-binding site of the kinase domain, is transmitted by the father to the two sons affected by persistent Müllerian duct syndrome, indicating a recessive autosomal transmission as in other cases of persistent Müllerian duct syndrome. Truncating mutations in receptors of the TGFbeta family exert dominant negative activity, which was seen only when each of the mutant anti-Müllerian hormone receptors was overexpressed in an anti-Müllerian hormone-responsive cell line. We conclude that assessment of dominant activity in vitro, which usually involves overexpression of mutant genes, does not necessarily produce information applicable to clinical conditions, in which mutant and endogenous genes are expressed on a one to one basis.     

“I am aware of the risks,I am not changing my behaviour”: risky sexual behaviour of university students in a high-HIV context

Unprotected sexual activity increases the risk of pregnancy and HIV and AIDS. More than three decades into the AIDS pandemic, the condom remains the most effective strategy for protecting against the dual risks of pregnancy and HIV and AIDS, but data from national surveys suggest that condom use among young people aged 15–24 is on the decline in South Africa. This study uses qualitative data from 20 in-depth interviews and one focus group to examine the risk behaviours of university students aged 18–24 years old, with particular emphasis on understanding the decline in the use of condoms. It is well documented that South Africa has one of the highest prevalence rates of HIV and AIDS in the world, however the findings of this study suggest that even though students were well informed about the perceived risks associated with unsafe sexual behaviours, they continue to engage in risk behaviours including unprotected sexual intercourse, multiple sexual partners and the use of alcohol. Male students were more likely than female students to report risky sexual behaviours. Condom use is occurring but not consistently. The majority of students did not use condoms during their first sexual encounter because of a lack of preparedness. Negative attitudes towards condoms continue to discourage students from using them consistently, especially those provided by the government. In light of this, it is recommended that rigorous efforts are directed towards challenging inaccurate perceptions about, and attitudes towards, condom use to promote consistency.     

Bacteraemia due to different groups of β-haemolytic streptococci: A two-year survey and presentation of a case of recurring infection due toStreptococcus “equisimilis”

Summary We present a 2-year survey of bacteraemic episodes due to -haemolytic streptococci and a case of recurring infection due to group C streptococcus,Streptococcus equisimilis. We found 53 episodes of bacteraemia with -haemolytic streptococci. Group A was the most common, followed by groups B, G and C. The proportion of nosocomial cases was the same in all four groups i. e. 24% (neonatal cases excluded). The clinical picture presented by groups C and G streptococcal cases was indistinguishable from that caused by group A streptococci, but patients with group G bacteraemia were older than patients with group A bacteraemia. Obvious clinical foci were more common in group A than group G cases. Disregarding neonatal cases, most patients had predisposing conditions. There was no difference in foci of bacteraemia, predisposing factors, treatment and outcome of disease. The overall mortality was 25%.

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Bakteriämie durch -hämolysierende Streptokokken: Zweijahresübersicht und Vorstellung eines Falles von rezidivierender Infektion durch Streptococcus equisimilis

Zusammenfassung Wir geben eine 2-Jahres-Übersicht über bakteriämische Episoden durch -hämolysierende Streptokokken und stellen einen Fall von rezidivierenden Infektionen durch Streptokokken der Gruppe C,Streptococcus equisimilis, vor. Wir fanden 53 Episoden von Bakteriämie durch -hämolysierende Streptokokken. Gruppe A war am häufigsten vertreten, es folgten die Gruppen B, G und C. Der Anteil nosokomialer Infektionen war in allen vier Gruppen gleich, das heißt 24% (Neugeborene ausgeschlossen). Das klinische Bild der durch Streptokokken der Gruppen C und G verursachten Infektionen war von den durch A Streptokokken verursachten nicht zu unterscheiden, doch waren Patienten, die durch Streptokokken der Gruppe G infiziert wurden, älter als Patienten mit Bakteriämie durch Streptokokken der Gruppe A. Bei Streptokokken der Gruppe A ließen sich häufiger eindeutige klinische Infektionsherde identifizieren als bei der Gruppe G. Abgesehen von den neonatalen Fällen hatten die meisten Patienten disponierende Faktoren. Hinsichtlich der Bakteriämieherde, disponierenden Faktoren, Behandlung und Krankheitsverlauf fanden sich keine Unterschiede. Die Gesamtsterblichkeit lag bei 25%.

     

“ … it’s almost therapeutic,right? Because it’s almost like that session that I never had”: gay men’s accounts of being a participant in HIV research

Limited research has explored how gay, bisexual and other men who have sex with men describe the impact of their involvement in HIV and sexual health research. We enrolled 166 gay and bisexual men who tested HIV-negative at a community sexual health clinic in Vancouver, British Columbia, into a year-long mixed methods study. Thirty-three of these participants who reported recent condomless anal intercourse were purposively recruited into an embedded qualitative study. Analysis revealed rich accounts of the self-described, interrelated impacts of study participation: (1) pride in contribution and community involvement (e.g., as a rationale for enrolment and an outcome of participation); (2) how one thinks about sexual behaviours and partnerships (e.g., encouraging reflection on the types and amount of sex they have had; in some cases the methods of quantitative data collection were said to have produced feelings of guilt or shame); and (3) experiencing research as a form of counselling (e.g., qualitative interviews were experienced as having a major therapeutic component to them). Our analysis underscores the importance of researchers being reflexive regarding how study participation in HIV research may impact participants, including unintended emotional and behavioural impacts.     

“Knowing I can be helpful makes me feel good inside,it makes me feel essential”: community health care workers’ experiences of conducting a home-based rehabilitation intervention for people living with HIV in KwaZulu-Natal,South Africa

People living with HIV (PLHIV) are living longer lives on antiretroviral therapy and are prone to a wide range of disabilities. Innovative strategies are required to meet the rehabilitation needs of PLHIV, particularly in resource-poor communities where HIV is endemic and access to institution-based rehabilitation is limited. Home-based rehabilitation (HBR) is one such approach, but there is a paucity of research related to HBR programmes for PLHIV or the experiences of community care workers (CCWs) involved in these programmes. Following a four month randomised controlled trial of a HBR intervention designed specifically for PLHIV in KwaZulu-Natal, South Africa; four CCWs were interviewed. This study employed a qualitative research design, using semi-structured interviews to explore these workers’ experiences of being involved in carrying out this intervention. Participants reported how their personal development, improvement in their own health and increased feelings of self-worth enabled them to successfully implement the intervention. Participants also described a number of inhibitors, including stigma and environmental challenges related to the distances between patients’ homes, the steep terrain and the hot climate. Despite this, the participants felt empowered by acquiring knowledge and skills that enabled them to shift roles beyond rehabilitation provision. The findings of this study should be considered when employing a task shifting approach in the development and implementation of HBR interventions for PLHIV. By employing a less specialised cadre of community workers to conduct basic HBR interventions, both the relative lack of qualified rehabilitation professionals and the high levels of disability in HIV-epidemic communities can be simultaneously addressed.