Arnon Henn

The ATPase cycle mechanism of the DEAD-box rRNA helicase, DbpA.

DEAD-box proteins are ATPase enzymes that destabilize and unwind duplex RNA. Quantitative knowledge of the ATPase cycle parameters is critical for developing models of helicase activity. However, limited information regarding the rate and equilibrium constants defining the ATPase cycle of RNA helicases is available, including the distribution of populated biochemical intermediates, the catalytic step(s) that limits the enzymatic reaction cycle, and how ATP utilization and RNA interactions are linked. We present a quantitative kinetic and equilibrium characterization of the ribosomal RNA (rRNA)-activated ATPase cycle mechanism of DbpA, a DEAD-box rRNA helicase implicated in ribosome biogenesis. rRNA activates the ATPase activity of DbpA by promoting a conformational change after ATP binding that is associated with hydrolysis. Chemical cleavage of bound ATP is reversible and occurs via a gamma-phosphate attack mechanism. ADP-P(i) and RNA binding display strong thermodynamic coupling, which causes DbpA-ADP-P(i) to bind rRNA with >10-fold higher affinity than with bound ATP, ADP or in the absence of nucleotide. The rRNA-activated steady-state ATPase cycle of DbpA is limited both by ATP hydrolysis and by P(i) release, which occur with comparable rates. Consequently, the predominantly populated biochemical states during steady-state cycling are the ATP- and ADP-P(i)-bound intermediates. Thermodynamic linkage analysis of the ATPase cycle transitions favors a model in which rRNA duplex destabilization is linked to strong rRNA and nucleotide binding. The presented analysis of the DbpA ATPase cycle reaction mechanism provides a rigorous kinetic and thermodynamic foundation for developing testable hypotheses regarding the functions and molecular mechanisms of DEAD-box helicases.

Cofilin-Linked Changes in Actin Filament Flexibility Promote Severing

The actin regulatory protein, cofilin, increases the bending and twisting elasticity of actin filaments and severs them. It has been proposed that filaments partially decorated with cofilin accumulate stress from thermally driven shape fluctuations at bare (stiff) and decorated (compliant) boundaries, thereby promoting severing. This mechanics-based severing model predicts that changes in actin filament compliance due to cofilin binding affect severing activity. Here, we test this prediction by evaluating how the severing activities of vertebrate and yeast cofilactin scale with the flexural rigidities determined from analysis of shape fluctuations. Yeast actin filaments are more compliant in bending than vertebrate actin filaments. Severing activities of cofilactin isoforms correlate with changes in filament flexibility. Vertebrate cofilin binds but does not increase the yeast actin filament flexibility, and does not sever them. Imaging of filament thermal fluctuations reveals that severing events are associated with local bending and fragmentation when deformations attain a critical angle. The critical severing angle at boundaries between bare and cofilin-decorated segments is smaller than in bare or fully decorated filaments. These measurements support a cofilin-severing mechanism in which mechanical asymmetry promotes local stress accumulation and fragmentation at boundaries of bare and cofilin-decorated segments, analogous to failure of some nonprotein materials.

Pathway of ATP utilization and duplex rRNA unwinding by the DEAD-box helicase, DbpA.

DEAD-box RNA helicase proteins use the energy of ATP hydrolysis to drive the unwinding of duplex RNA. However, the mechanism that couples ATP utilization to duplex RNA unwinding is unknown. We measured ATP utilization and duplex RNA unwinding by DbpA, a non-processive bacterial DEAD-box RNA helicase specifically activated by the peptidyl transferase center (PTC) of 23S rRNA. Consumption of a single ATP molecule is sufficient to unwind and displace an 8 base pair rRNA strand annealed to a 32 base pair PTC-RNA “mother strand” fragment. Strand displacement occurs after ATP binding and hydrolysis but before Pi product release. Pi release weakens binding to rRNA, thereby facilitating the release of the unwound rRNA mother strand and the recycling of DbpA for additional rounds of unwinding. This work explains how ATPase activity of DEAD-box helicases is linked to RNA unwinding.

Vertebrate Myosin VIIb Is a High Duty Ratio Motor Adapted for Generating and Maintaining Tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (∼1 s-1), activated by very low actin filament concentrations (KATPase ∼ 0.7 μm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin·ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 μm actin, the duty ratio of myosin VIIb is ∼0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.

ATP Utilization and RNA Conformational Rearrangement by DEAD-Box Proteins

RNA helicase enzymes catalyze the in vivo folding and conformational re-arrangement of RNA. DEAD-box proteins (DBPs) make up the largest family of RNA helicases and are found across all phyla. DBPs are molecular motor proteins that utilize chemical energy in cycles of ATP binding, hydrolysis, and product release to perform mechanical work resulting in reorganization of cellular RNAs. DBPs contain a highly conserved motor domain helicase core. Auxiliary domains, enzymatic adaptations, and regulatory partner proteins contribute to the diversity of DBP function throughout RNA metabolism. In this review we focus on the current understanding of the DBP ATP utilization mechanism in rearranging and unwinding RNA structures. We discuss DBP structural properties, kinetic pathways, and thermodynamic features of nucleotide-dependent interactions with RNA. We highlight recent advances in the DBP field derived from biochemical and molecular biophysical investigations aimed at developing a quantitative mechanistic understanding of DBP molecular motor function.

Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins.

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (P(i)) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-P(i) binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116-ADP-P(i) state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with P(i) release dissociates Mss116-RNA complexes, regenerating free Mss116.

Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.

Myosin Isoform Determines the Conformational Dynamics and Cooperativity of Actin Filaments in the Strongly Bound Actomyosin Complex

We used transient phosphorescence anisotropy to detect the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin strongly bound to single-headed fragments of muscle myosin subfragment 1 (S1) and non-muscle myosin V (MV). The conformational dynamics of actin filaments in solution are markedly influenced by the isoform of bound myosin. Both myosins increase the final anisotropy of actin at substoichiometric binding densities, indicating long-range, non-nearest neighbor cooperative restriction of filament rotational dynamics amplitude, but the cooperative unit is larger with MV than with muscle S1. Both myosin isoforms also cooperatively affect the actin filament rotational correlation time, but with opposite effects: muscle S1 decreases rates of intrafilament torsional motion, while binding of MV increases the rates of motion. The cooperative effects on the rates of intrafilament motions correlate with the kinetics of myosin binding to actin filaments such that MV binds more rapidly and muscle myosin binds more slowly to partially decorated filaments than to bare filaments. The two isoforms also differ in their effects on the phosphorescence lifetime of the actin-bound erythrosin iodoacetamide: while muscle S1 increases the lifetime, suggesting decreased aqueous exposure of the probe, MV does not induce a significant change. We conclude that the dynamics and structure of actin in the strongly bound actomyosin complex are determined by the isoform of the bound myosin in a manner likely to accommodate the diverse functional roles of actomyosin in muscle and non-muscle cells.

Myo19 is an outer mitochondrial membrane motor and effector of starvation-induced filopodia

ABSTRACT Mitochondria respond to environmental cues and stress conditions. Additionally, the disruption of the mitochondrial network dynamics and its distribution is implicated in a variety of neurodegenerative diseases. Here, we reveal a new function for Myo19 in mitochondrial dynamics and localization during the cellular response to glucose starvation. Ectopically expressed Myo19 localized with mitochondria to the tips of starvation-induced filopodia. Corollary to this, RNA interference (RNAi)-mediated knockdown of Myo19 diminished filopodia formation without evident effects on the mitochondrial network. We analyzed the Myo19–mitochondria interaction, and demonstrated that Myo19 is uniquely anchored to the outer mitochondrial membrane (OMM) through a 30–45-residue motif, indicating that Myo19 is a stably attached OMM molecular motor. Our work reveals a new function for Myo19 in mitochondrial positioning under stress. Summary: Myo19 is an effector of starvation-induced filopodia formation and is directly anchored to the outer mitochondrial membrane.

RNA-dependent chromatin localization of KDM4D lysine demethylase promotes H3K9me3 demethylation

The JmjC-containing lysine demethylase, KDM4D, demethylates di-and tri-methylation of histone H3 on lysine 9 (H3K9me3). How KDM4D is recruited to chromatin and recognizes its histone substrates remains unknown. Here, we show that KDM4D binds RNA independently of its demethylase activity. We mapped two non-canonical RNA binding domains: the first is within the N-terminal spanning amino acids 115 to 236, and the second is within the C-terminal spanning amino acids 348 to 523 of KDM4D. We also demonstrate that RNA interactions with KDM4D N-terminal region are critical for its association with chromatin and subsequently for demethylating H3K9me3 in cells. This study implicates, for the first time, RNA molecules in regulating the levels of H3K9 methylation by affecting KDM4D association with chromatin.