Enteroviral 2C protein is an RNA-stimulated ATPase and uses a two-step mechanism for binding to RNA and ATP.

The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here, we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state. 2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the α and ß phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase.

Two Distinct Modes of DNA Binding by an MCM Helicase Enable DNA Translocation.

A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism Saccharolobus solfataricus binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation.

MCM ring hexamerization is a prerequisite for DNA-binding.

The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings by showing that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.

Dynamic look at DNA unwinding by a replicative helicase.

A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.

Fundamental Characteristics of AAA+ Protein Family Structure and Function.

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

Archaeal MCM Proteins as an Analog for the Eukaryotic Mcm2-7 Helicase to Reveal Essential Features of Structure and Function.

In eukaryotes, the replicative helicase is the large multisubunit CMG complex consisting of the Mcm2-7 hexameric ring, Cdc45, and the tetrameric GINS complex. The Mcm2-7 ring assembles from six different, related proteins and forms the core of this complex. In archaea, a homologous MCM hexameric ring functions as the replicative helicase at the replication fork. Archaeal MCM proteins form thermostable homohexamers, facilitating their use as models of the eukaryotic Mcm2-7 helicase. Here we review archaeal MCM helicase structure and function and how the archaeal findings relate to the eukaryotic Mcm2-7 ring.

Rare SNP in the HELB gene interferes with RPA interaction and cellular function of HELB.

HELB is a human helicase involved in initiation of DNA replication, the replication stress response, and regulation of double-strand DNA break repair. rs75770066 is a rare SNP in the HELB gene that affects age at natural menopause. rs75770066 results in a D506G substitution in an acidic patch within the 1A domain of the helicase that is known to interact with RPA. We found that this amino acid change dramatically impairs the cellular function of HELB. D506G-HELB exhibits impaired interaction with RPA, which likely results in the effects of rs75770066 as this reduces recruitment of HELB to sites of DNA damage. Reduced recruitment of D506G-HELB to double-strand DNA breaks and the concomitant increase in homologous recombination likely alters the levels of meiotic recombination, which affects the viability of gametes. Because menopause occurs when oocyte levels drop below a minimum threshold, altered repair of meiotic double-stranded DNA breaks has the potential to directly affect the age at natural menopause.

Mechanism of DNA translocation in a replicative hexameric helicase.

The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.

On helicases and other motor proteins.

Helicases are molecular machines that utilize energy derived from ATP hydrolysis to move along nucleic acids and to separate base-paired nucleotides. The movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. Recent structural data for the hexameric E1 helicase of papillomavirus in complex with single-stranded DNA and MgADP has provided a detailed atomic and mechanistic picture of its ATP-driven DNA translocation. The structural and mechanistic features of this helicase are compared with the hexameric helicase prototypes T7gp4 and SV40 T-antigen. The ATP-binding site architectures of these proteins are structurally similar to the sites of other prototypical ATP-driven motors such as F1-ATPase, suggesting related roles for the individual site residues in the ATPase activity.

Structure of a dimer of the Sulfolobus solfataricus MCM N-terminal domain reveals a potential role in MCM ring opening.

Cells strongly regulate DNA replication to ensure genomic stability and prevent several diseases, including cancers. Eukaryotes and archaea strictly control DNA-replication initiation by the regulated loading of hexameric minichromosome maintenance (MCM) rings to encircle both strands of the DNA double helix followed by regulated activation of the loaded rings such that they then encircle one DNA strand while excluding the other. Both steps involve an open/closed ring transformation, allowing DNA strands to enter or exit. Here, the crystal structure of a dimer of the N-terminal domain of Sulfolobus solfataricus MCM with an intersubunit interface that is more extensive than in closed-ring structures, while including common interactions to enable facile interconversion, is presented. It is shown that the identified interface could stabilize open MCM rings by compensating for lost interactions at an open neighbor interface and that the prior open-ring cryo-EM structure of MCM loading has a similar extended interface adjacent to its open interface.

DNA translocation mechanism of the MCM complex and implications for replication initiation.

The DNA translocation activity of the minichromosome maintenance (MCM) complex powers DNA strand separation of the replication forks of eukaryotes and archaea. Here we illustrate an atomic level mechanism for this activity with a crystal structure of an archaeal MCM hexamer bound to single-stranded DNA and nucleotide cofactors. Sequence conservation indicates this rotary mechanism is fully possible for all eukaryotes and archaea. The structure definitively demonstrates the ring orients during translocation with the N-terminal domain leading, indicating that the translocation activity could also provide the physical basis of replication initiation where a double-hexamer idly encircling double-stranded DNA transforms to single-hexamers that encircle only one strand. In this mechanism, each strand binds to the N-terminal tier of one hexamer and the AAA+ tier of the other hexamer such that one ring pulls on the other, aligning equivalent interfaces to enable each hexamer to pull its translocation strand outside of the opposing hexamer.

Genomic subtyping and therapeutic targeting of acute erythroleukemia.

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosis in the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53 mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis and TP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition. This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia.

Structure of a double hexamer of the Pyrococcus furiosus minichromosome maintenance protein N-terminal domain.

The crystal structure of the N-terminal domain of the Pyrococcus furiosus minichromosome maintenance (MCM) protein as a double hexamer is described. The MCM complex is a ring-shaped helicase that unwinds DNA at the replication fork of eukaryotes and archaea. Prior to replication initiation, the MCM complex assembles as an inactive double hexamer at specific sites of DNA. The presented structure is highly consistent with previous MCM double-hexamer structures and shows two MCM hexamers with a head-to-head interaction mediated by the N-terminal domain. Minor differences include a diminished head-to-head interaction and a slightly reduced inter-hexamer rotation.

Cancer-associated mutants of RNA helicase DDX3X are defective in RNA-stimulated ATP hydrolysis.

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension ("ATP-binding loop", ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures suggesting that the ABL interacts dynamically with ATP and confirming that the interaction occurs in solution by NMR chemical shift perturbation and isothermal titration calorimetry. DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR chemical shift perturbation to show that DDX3X interacts specifically with double-stranded RNA through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of Schizosaccharomyces pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. Taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.

Stereospecificity and Self-Selectivity in the Generation of a Chiral Molecular Tetrahedron by Metal-Assisted Self-Assembly.

The chiral bidentate ligand (S,S)-1 reacts stereospecifically with Ga3+ to generate a [Ga4 (L)6 ]12- molecular tetrahedron although similar ligands generate [Ga2 (L)3 ]6- complexes. The assembly of this complex is self-selective as a racemic mixture of the ligand sorts by chirality to generate an enantiomeric pair of homochiral complexes.

Ligand Self-Recognition in the Self-Assembly of a [{Cu(L)}2 ]2+ Complex: The Role of Chirality.

The chirality alone of a conformationally restricted, bifunctional ligand (L) is the basis for the self-recognition process schematically represented below. A racemic mixture of these ligands reacts with Cu+ ions quantitatively to generate a racemic mixture of a [(CuL)2 ]2+ homochiral complex (represented by cubes), where each complex contains ligands with identical configurations.

Analysis of the crystal structure of an active MCM hexamer.

In a previous Research article (Froelich et al., 2014), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

A conserved MCM single-stranded DNA binding element is essential for replication initiation.

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

Emergence of polyclonal FLT3 tyrosine kinase domain mutations during sequential therapy with sorafenib and sunitinib in FLT3-ITD-positive acute myeloid leukemia.

PURPOSE: To evaluate the clinical activity of sequential therapy with sorafenib and sunitinib in FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD)-positive acute myelogenous leukemia (AML) and monitor the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations during treatment. EXPERIMENTAL DESIGN: Six children with relapsed/refractory AML were treated with sorafenib in combination with clofarabine and cytarabine, followed by single-agent sorafenib if not a candidate for transplantation. Sunitinib was initiated after sorafenib relapse. Bone marrow samples were obtained for assessment of FLT3 TKD mutations by deep amplicon sequencing. The phase of secondary mutations with ITD alleles was assessed by cloning and sequencing of FLT3 exons 14 through 20. Identified mutations were modeled in Ba/F3 cells, and the effect of kinase inhibitors on FLT3 signaling and cell viability was assessed. RESULTS: Four patients achieved complete remission, but 3 receiving maintenance therapy with sorafenib relapsed after 14 to 37 weeks. Sunitinib reduced circulating blasts in two patients and marrow blasts in one. Two patients did not respond to sorafenib combination therapy or sunitinib. FLT3 mutations at residues D835 and F691 were observed in sorafenib resistance samples on both ITD-positive and -negative alleles. Deep sequencing revealed low-level mutations and their evolution during sorafenib treatment. Sunitinib suppressed leukemic clones with D835H and F691L mutations, but not D835Y. Cells expressing sorafenib-resistant FLT3 mutations were sensitive to sunitinib in vitro. CONCLUSIONS: Sunitinib has activity in patients that are resistant to sorafenib and harbor secondary FLT3 TKD mutations. The use of sensitive methods to monitor FLT3 mutations during therapy may allow individualized treatment with the currently available kinase inhibitors.

Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum.

Erythrocyte binding antigen 175 (EBA-175) is a P. falciparum protein that binds the major glycoprotein found on human erythrocytes, glycophorin A, during invasion. Here we present the crystal structure of the erythrocyte binding domain of EBA-175, RII, which has been established as a vaccine candidate. Binding sites for the heavily sialylated receptor glycophorin A are proposed based on a complex of RII with a glycan that contains the essential components required for binding. The dimeric organization of RII displays two prominent channels that contain four of the six observed glycan binding sites. Each monomer consists of two Duffy binding-like (DBL) domains (F1 and F2). F2 more prominently lines the channels and makes the majority of the glycan contacts, underscoring its role in cytoadherence and in antigenic variation in malaria. Our studies provide insight into the mechanism of erythrocyte invasion by the malaria parasite and aid in rational drug design and vaccines.