Probing Motions between Equivalent RNA Domains Using Magnetic Field Induced Residual Dipolar Couplings:  Accounting for Correlations between Motions and Alignment

Zhang, Qi; Throolin, Rachel; Pitt, Stephen W.; Serganov, Alexander; Al‐Hashimi, Hashim M.

doi:10.1021/ja0363056

Cited by 60 publications

(98 citation statements)

References 19 publications

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“…We have recently obtained independent support for such large amplitude global motions in TAR-FREE using direct magnetic field magneticinduced RDCs. 46 Remarkably, the stem-specific ϑ values (Table 1) determined here for TAR-ARG are in far better agreement (ϑ stemI = (1.26 ± 0.04) × 10 −3 and ϑ stemII = (1.36 ± 0.03) × 10 −3 ). The resulting ϑ int value of 0.93(± 0.07) (Table 1) approaches unity, consistent with a rigid inter-helical conformation (Figure 1(e)).…”

Section: Author Manuscript Author Manuscriptsupporting

confidence: 47%

“…We have recently obtained independent support for such large amplitude global motions in TAR-FREE using direct magnetic field magneticinduced RDCs. 46 Remarkably, the stem-specific ϑ values (Table 1) (Table 1) approaches unity, consistent with a rigid inter-helical conformation (Figure 1(e)). Hence, as is the case for Mg 2+ ( Figure 1(e)), ARG binding dramatically stabilizes the global conformation of TAR.…”

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confidence: 54%

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Argininamide Binding Arrests Global Motions in HIV-1 TAR RNA: Comparison with Mg2+-induced Conformational Stabilization

Pitt

Majumdar

Serganov

et al. 2004

Journal of Molecular Biology

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122

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The structure and dynamics of the stem-loop transactivation response element (TAR) RNA from the human immunodeficiency virus type-1 (HIV-1) bound to the ligand argininamide (ARG) has been characterized using a combination of a large number of residual dipolar couplings (RDCs) and trans-hydrogen bond NMR methodology. Binding of ARG to TAR changes the average interhelical angle between the two stems from ~47° in the free state to ~11° in the bound state, and leads to the arrest of large amplitude (±46°) inter-helical motions observed previously in the free state. While the global structural dynamics of TAR-ARG is similar to that previously reported for TAR bound to Mg 2+ , there are substantial differences in the hydrogen bond alignment of bulge and neighboring residues. Based on a novel H5(C5)NN experiment for probing hydrogenmediated 2h J(N,N) scalar couplings as well as measured RDCs, the TAR-ARG complex is stabilized by a U38-A27·U23 base-triple involving an A27·U23 reverse Hoogsteen hydrogen bond alignment as well as by a A22-U40 Watson-Crick base-pair at the junction of stem I. These hydrogen bond alignments are not observed in either the free or Mg 2+ bound forms of TAR. The combined conformational analysis of TAR under three states reveals that ligands and divalent ions can stabilize similar RNA global conformations through distinct interactions involving different hydrogen bond alignments in the RNA. Keywords recognition; adaptation; collective motions; NMR; residual dipolar couplingsThere is now ample evidence indicating that many RNAs do not fold into a single welldefined structure, but rather exist as an ensemble of interconverting conformations. [21][22][23] By being uniquely sensitive to motional averaging over a wide window of time scales (picoseconds-milliseconds), the measurement of RDCs has also emerged as a powerful approach for probing the amplitudes and directions of collective motions in biomolecules. [24][25][26][27] We recently employed RDCs to investigate the conformational dynamics of human immunodefiency virus type 1 (HIV-1) transactivation response element (TAR) RNA ( Figure 1(a)). 28,29 The TAR domain has been the subject of numerous investigations because its interaction with the transactivator protein (Tat) is critical for HIV-1 viral replication, 30 rendering it a potential target for therapeutic development. 31 Previous structural studies had established that binding to peptide mimics of Tat, including the ligand argininamide (ARG), induces a change in the TAR global conformation 32-37 from a bent to coaxial interhelical alignment. However little information was available regarding global motions in TAR and its potential role in recognition. Our RDC-NMR study of TAR in the divalent ion free state (TAR-FREE) 28 provided evidence that the two helices undergo large amplitude (±46°) rigid-body collective motions about an average inter-helical angle of 47°. Because the coaxially aligned bound TAR conformations appeared to be dynamically accessible in the free state, these resu...

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Section: Author Manuscript Author Manuscriptsupporting

confidence: 47%

“…We have recently obtained independent support for such large amplitude global motions in TAR-FREE using direct magnetic field magneticinduced RDCs. 46 Remarkably, the stem-specific ϑ values (Table 1) (Table 1) approaches unity, consistent with a rigid inter-helical conformation (Figure 1(e)). Hence, as is the case for Mg 2+ ( Figure 1(e)), ARG binding dramatically stabilizes the global conformation of TAR.…”

mentioning

confidence: 54%

Argininamide Binding Arrests Global Motions in HIV-1 TAR RNA: Comparison with Mg2+-induced Conformational Stabilization

Pitt

Majumdar

Serganov

et al. 2004

Journal of Molecular Biology

Self Cite

122

View full text Add to dashboard Cite

show abstract

“…Here, the derived motional amplitudes will underestimate the real motional amplitudes 24 . Simulations (data not shown) as well as experimental results show that differences on the order of three base pairs can be sufficient to take an RNA system outside the extreme coupling limit and into the intermediate regime 21,22,24,38,54,55 . Extensive elongation of helices using isotopic labeling strategies that render elongation residues invisible can also be used to bring the RNA system closer to within the decoupling limit 56 .…”

Section: Experimental Designmentioning

confidence: 98%

“…Second, the order tensor analysis of RDCs assumes that one fragment dominates overall molecular alignment of the RNA 24,47,53,54 . This "decoupling limit" (i.e., when individual helix motions are decoupled from global motions) is readily satisfied when one helix dominates alignment or when helices are held rigid relative to one another.…”

Section: Experimental Designmentioning

confidence: 99%

“…In the extreme coupling limit, helices have similar size and shape and contribute equally to total alignment. If motions of the helices-one relative to the other-result in equivalent changes in total alignment, then similar degrees of helix order will be observed regardless of interhelical motions 54 . In such cases, observation of ϑ int values equal to one does not rule out the presence of interhelix motions.…”

Section: Experimental Designmentioning

confidence: 99%

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Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

et al. 2007

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We present a protocol for determining the relative orientation and dynamics of A-form helices in 13 C/ 15 N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson-Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.

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Evidence that Electrostatic Interactions Dictate the Ligand‐Induced Arrest of RNA Global Flexibility

Pitt¹,

Zhang²,

Patel³

et al. 2005

Angewandte Chemie

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Keywords conformation analysis; drug design; NMR spectroscopy; RNA Arresting flexibility to prevent RNA from undergoing functionally important conformational transitions is an established strategy for developing RNA-targeting therapeutics. [1] To understand the role of RNA flexibility in adaptive recognition [2] is also important for the rational design of small molecules that bind their RNA target with high affinity and specificity. [3] Yet few studies have quantitatively examined how RNA-binding therapeutics affect the flexibility of their RNA targets. As a result, little is known about the RNA-ligand interactions that are important for arresting different types of RNA functional flexibility. Here, we provide direct evidence by using NMR residual dipolar couplings (RDCs) [4] that electrostatic interactions play a primary role in dictating the degree to which small molecules arrest global motions in RNA.

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Probing Motions between Equivalent RNA Domains Using Magnetic Field Induced Residual Dipolar Couplings: Accounting for Correlations between Motions and Alignment

Cited by 60 publications

References 19 publications

Argininamide Binding Arrests Global Motions in HIV-1 TAR RNA: Comparison with Mg2+-induced Conformational Stabilization

Argininamide Binding Arrests Global Motions in HIV-1 TAR RNA: Comparison with Mg2+-induced Conformational Stabilization

Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings

Evidence that Electrostatic Interactions Dictate the Ligand‐Induced Arrest of RNA Global Flexibility

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