Ivan A. Belashov

Core-binding factor β increases the affinity between human Cullin 5 and HIV-1 Vif within an E3 ligase complex.

HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor β (CBFβ) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif((1-192))/EloB/C/CBFβ (K(d) = 5 ± 2 nM) than to Vif((95-192))/EloB/C (K(d) = 327 ± 40 nM), which cannot bind CBFβ. A comparison of heat capacity changes supports a model in which CBFβ prestabilizes Vif((1-192)) relative to Vif((95-192)), consistent with a stronger interaction of Cul5 with Vifs C-terminal Zn(2+)-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications.

Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Significance Riboswitches are RNA molecules found mostly in bacteria that control genes by sensing cellular levels of metabolites, such as the simple organic compound preQ1. The diversity of riboswitches and their potential as novel antibiotic targets continue to elicit interest in these regulatory sequences. Here we present the crystal structure of a newly discovered bacterial preQ1-III riboswitch that senses preQ1 using an unusual, two-part architecture. A complementary analysis of flexibility and dynamics showed that recognition of preQ1 induces riboswitch compaction, while concomitantly enhancing formation of a distant double-helix possessing a regulatory signal that zips and unzips rapidly, producing gene “off” and “on” states. These observations expand our knowledge of riboswitch construction and suggest a broader role for dynamics than previously recognized. PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3′ RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1–P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼0.6 s−1) and undocking (kundock ∼1.1 s−1). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

Metalloriboswitches: RNA-Based Inorganic Ion Sensors that Regulate Genes

Divalent ions fulfill essential cellular roles and are required for virulence by certain bacteria. Free intracellular Mg2+ can approach 5 mm, but at this level Mn2+, Ni2+, or Co2+ can be growth-inhibitory, and magnesium fluoride is toxic. To maintain ion homeostasis, many bacteria have evolved ion sensors embedded in the 5′-leader sequences of mRNAs encoding ion uptake or efflux channels. Here, we review current insights into these “metalloriboswitches,” emphasizing ion-specific binding by structured RNA aptamers and associated conformational changes in downstream signal sequences. This riboswitch-effector interplay produces a layer of gene regulatory feedback that has elicited interest as an antibacterial target.

Observation of preQ1-II riboswitch dynamics using single-molecule FRET

ABSTRACT PreQ1 riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ1. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ1-bound states of the preQ1-II riboswitch from Lactobacillus rhamnosus. We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ1 stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ1-II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.

Structure of HIV TAR in complex with a Lab-Evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription

Abstract Natural and lab-evolved proteins often recognize their RNA partners with exquisite affinity. Structural analysis of such complexes can offer valuable insight into sequence-selective recognition that can be exploited to alter biological function. Here, we describe the structure of a lab-evolved RNA recognition motif (RRM) bound to the HIV-1 trans-activation response (TAR) RNA element at 1.80 Å-resolution. The complex reveals a trio of arginines in an evolved β2–β3 loop penetrating deeply into the major groove to read conserved guanines while simultaneously forming cation-π and salt-bridge contacts. The observation that the evolved RRM engages TAR within a double-stranded stem is atypical compared to most RRMs. Mutagenesis, thermodynamic analysis and molecular dynamics validate the atypical binding mode and quantify molecular contributions that support the exceptionally tight binding of the TAR-protein complex (KD,App of 2.5 ± 0.1 nM). These findings led to the hypothesis that the β2–β3 loop can function as a standalone TAR-recognition module. Indeed, short constrained peptides comprising the β2–β3 loop still bind TAR (KD,App of 1.8 ± 0.5 μM) and significantly weaken TAR-dependent transcription. Our results provide a detailed understanding of TAR molecular recognition and reveal that a lab-evolved protein can be reduced to a minimal RNA-binding peptide.