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. 2010 Dec 23;5(12):e15675.
doi: 10.1371/journal.pone.0015675.

Structural basis of the association of HIV-1 matrix protein with DNA

Mengli Cai  1 , Ying Huang, Robert Craigie, G Marius Clore
Affiliations

Structural basis of the association of HIV-1 matrix protein with DNA

Mengli Cai et al. PLoS One. .

Abstract

HIV-1 matrix (MA) is a multifunctional protein that is synthesized as a polyprotein that is cleaved by protease during viral maturation. MA contains a cluster of basic residues whose role is controversial. Proposed functions include membrane anchoring, facilitating viral assembly, and directing nuclear import of the viral DNA. Since MA has been reported to be a component of the preintegration complex (PIC), we have used NMR to probe its interaction with other PIC components. We show that MA interacts with DNA and this is likely sufficient to account for its association with the PIC.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. DNA oligonucleotide duplexes used in the current study.
(A) and (B) are 12 mer and 16 mer DNA fragments used for chemical shift mapping and ITC measurements. DNA fragment (B) was also used for the RDC measurements. (C) and (D) are 16 mer DNA fragments containing dT-EDTA as indicated and were used for PRE measurements.
Figure 2
Figure 2. Interaction of MA with DNA monitored by 1H-15N HSQC spectroscopy.
(A) Selected region of the 1H-5N-edited HSQC spectrum (recorded at a spectrometer frequency of 500 MHz) of free 15N-labeled MA (left panel) and 15N-labeled MA complexed with 16 mer DNA. Cross-peaks that are either shifted or intensified upon complexation are labeled.
Figure 3
Figure 3. 1HN/15N chemical shift perturbation mapping and PRE measurements on the interaction of 15N-labeled MA with DNA.
(A) 1HN/15N chemical shift difference (ΔδH/N = (ΔδN2+ΔδHN 2)1/2] in Hz between the MA/DNA complex and free MA at a spectrometer frequency of 500 MHz. (B) and (C) 1HN-Γ2 PRE rates as a function of residue for 15N-labeled MA complexed to paramagnetically-labeled DNA; the oligonucleotides in (B) and (C) correspond to the oligonucleotides shown in Figs. 1C and D, respectively, with dT-EDTA-Mn2+ located at opposite ends of the DNA. The PRE data were recorded at a spectrometer frequency of 600 MHz.
Figure 4
Figure 4. ITC measurements of the interaction of MA with DNA.
12 mer DNA (left panel) and 16 mer DNA (right panel) duplexes shown in Figs. 1A and B.
Figure 5
Figure 5. Comparison of observed ([Image: see text]) RDCs measured for the MA/DNA complex with those calculated ([Image: see text]) from the crystal structure of free MA.
The dipolar coupling R-factor (defined as the ratio of the rms deviation between observed and calculated values and the expected rms deviation if the vectors were randomly distributed given by [Image: see text] , where Da is the magnitude of the principal component of the alignment tensor and η the rhombicity) is 21.9%. The X-ray coordinates were taken from (PDB code 1HIW), and addition of backbone amide protons and best-fitting of RDCs by singular value decomposition (SVD) was carried out using Xplor-NIH . The values of [Image: see text] and η are 5.6 Hz and 0.3, respectively. The RDC data were measured using 11 mg/ml phage pf1 at a spectrometer frequency of 800 MHz, and a large excess of DNA was employed to ensure that all MA present was bound to DNA and only one MA molecule was bound per DNA duplex.
Figure 6
Figure 6. Model of the non-specific MA/DNA complex built on the basis of 1HN/15N chemical shift perturbation, PRE and RDC measurements.
The chemical shift perturbation and PRE measurements delineate the DNA binding surface on MA, while the RDC data permit one to align MA relative to the long axis of the DNA. Alignment of protein/DNA complexes where the DNA is essentially undistorted B-form DNA is dominated by the electrostatic properties of the DNA and the principal (z) axis of the alignment tensor is known to be essentially parallel to the long axis of the DNA . The DNA is shown as a gold ribbon, MA as a red backbone tube with selected side chains as sticks, and the axes of the alignment tensor in black.
See this image and copyright information in PMC

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