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. 2014 Jul 16;9(7):e102150.
doi: 10.1371/journal.pone.0102150. eCollection 2014.

Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers

Loussiné Zargarian  1 , Carine Tisné  2 , Pierre Barraud  2 , Xiaoqian Xu  3 , Nelly Morellet  4 , Brigitte René  1 , Yves Mély  5 , Philippe Fossé  1 , Olivier Mauffret  1
Affiliations

Dynamics of linker residues modulate the nucleic acid binding properties of the HIV-1 nucleocapsid protein zinc fingers

Loussiné Zargarian et al. PLoS One. .

Abstract

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes established a new property: the unpaired guanines targeted by NC are more often inserted in the C-terminal zinc finger (ZF2) than in the N-terminal zinc finger (ZF1). Although previous NMR dynamic studies were performed with NC, the dynamic behavior of the linker residues connecting the two ZF domains remains unclear. This prompted us to investigate the dynamic behavior of the linker residues. Here, we collected 15N NMR relaxation data and used for the first time data at several fields to probe the protein dynamics. The analysis at two fields allows us to detect a slow motion occurring between the two domains around a hinge located in the linker at the G35 position. However, the amplitude of motion appears limited in our conditions. In addition, we showed that the neighboring linker residues R29, A30, P31, R32, K33 displayed restricted motion and numerous contacts with residues of ZF1. Our results are fully consistent with a model in which the ZF1-linker contacts prevent the ZF1 domain to interact with unpaired guanines, whereas the ZF2 domain is more accessible and competent to interact with unpaired guanines. In contrast, ZF1 with its large hydrophobic plateau is able to destabilize the double-stranded regions adjacent to the guanines bound by ZF2. The linker residues and the internal dynamics of NC regulate therefore the different functions of the two zinc fingers that are required for an optimal chaperone activity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A/Sequence of the HIV-1 nucleocapsid protein (NL4-3).
B/Experimental 15N NMR relaxation data ((a) Longitudinal (T1), (b) transverse (T2) relaxation times and (c) heteronuclear nOe) for backbone atoms obtained at 950 MHz for NC at 10°C.
Figure 2
Figure 2. Experimental T1/T2 (or T1/) ratios
obtained at 500 MHz (this work) (a), 600 MHz (b), 950 MHz (this work) (c) for NC at 10°C. At each field, the average value for each zinc finger (ZF1 and ZF2) is indicated as a red bold line. For ZF1, the values for residues 15 to 28 are considered, while for ZF2, residues 36 to 49 are considered.
Figure 3
Figure 3. S2 order parameters determined from Model-Free analysis of 15N relaxation data
obtained at 950°C.
Figure 4
Figure 4. Views of four NC-oligonucleotide complexes.
(a) SL3-NC (pdb 1a1t); (b) PBS-NC (2exf); (c) SL2-NC (1f6u); (d) mini-cTAR-NC (2l4l). For each complex, the oligonucleotide and protein backbones are underlined in orange and green, respectively. The protein side chains are not shown except for W37 (stick in violet) in ZF2, and V13, F16, A25, I/T 24 in ZF1 (spheres in yellow). Additionally, the stem residue closest to the loop is shown as a sphere (color mode by atom type). In (a) and (c), the guanine bound to ZF1 is indicated in stick and light grey
Figure 5
Figure 5. Schematic representation of the task specialization of each ZF upon binding to nucleic acids.
The nucleic acid fragment is in orange, NC zinc-fingers are in yellow (ZF1) and purple (ZF2), and the rest of the protein is in green. (a) Situation #1 with two accessible guanines. ZF2 binds first to an accessible guanine, and ZF1 binds the remaining guanine. The stem is not destabilized. (b) Situation #2 with only one accessible guanine. ZF2 binds first to this accessible guanine, and ZF1 remains free to contact the stem via its large hydrophobic platform. The stem is destabilized (in blue).
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Grants and funding

ANRS and CNRS supported the work. Financial support from the TGIR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged. The funders has no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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