Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan 25;1(1):66-78.
doi: 10.1021/jacsau.0c00039. Epub 2020 Dec 9.

Fuzzy Association of an Intrinsically Disordered Protein with Acidic Membranes

Alan Hicks  1   1   2 , Cristian A Escobar  1   3 , Timothy A Cross  1   1   3 , Huan-Xiang Zhou  2
Affiliations

Fuzzy Association of an Intrinsically Disordered Protein with Acidic Membranes

Alan Hicks et al. JACS Au. .

Abstract

Many physiological and pathophysiological processes, including Mycobacterium tuberculosis (Mtb) cell division, may involve fuzzy membrane association by proteins via intrinsically disordered regions. The fuzziness is extreme when the conformation and pose of the bound protein and the composition of the proximal lipids are all highly dynamic. Here, we tackled the challenge in characterizing the extreme fuzzy membrane association of the disordered, cytoplasmic N-terminal region (NT) of ChiZ, an Mtb divisome protein, by combining solution and solid-state NMR spectroscopy and molecular dynamics simulations. While membrane-associated NT does not gain any secondary structure, its interactions with lipids are not random, but formed largely by Arg residues predominantly in the second, conserved half of the NT sequence. As NT frolics on the membrane, lipids quickly redistribute, with acidic lipids, relative to zwitterionic lipids, preferentially taking up Arg-proximal positions. The asymmetric engagement of NT arises partly from competition between acidic lipids and acidic residues, all in the first half of NT, for Arg interactions. This asymmetry is accentuated by membrane insertion of the downstream transmembrane helix. This type of semispecific molecular recognition may be a general mechanism by which disordered proteins target membranes.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Sequence and structure of ChiZ. (a) Amino-acid sequence. Disordered regions, transmembrane helix, and LysM domain are indicated by black, green, and brown letters, respectively; acidic and Arg residues in disordered regions are shown as red and blue letters, respectively. (b) Disposition of different regions or domains with respect to the membrane. Colors match those in panel (a). The compositions of the three ChiZ constructs are also indicated.
Figure 2
Figure 2
Solution 15N–1H HSQC spectra of ChiZ1-64 in the presence and absence of liposomes. ChiZ1-64 spectra without and with liposomes are shown as black and red contours, respectively. ChiZ1-64 was mixed with (a) POPC, (b) DOPC:DOPE (4:1 molar ratio), (c) POPC:POPG (4:1 molar ratio), and (d) POPG:POPE (7:3 molar ratio) liposomes at a protein to lipid ratio of 1:100 in 20 mM phosphate buffer (pH 7.0) containing 25 mM NaCl. All spectra were collected with 100 μM protein at 25 °C.
Figure 3
Figure 3
Solid-state NMR data of ChiZ1-64 bound to and ChiZ-FL reconstituted into POPG:POPE liposomes. The protein to lipid ratios were 1:50 and 1:80, respectively, for the two constructs. (a) 13C–13C correlation spectra of ChiZ1-64 using INEPT and CP magnetization transfer, shown in black and red, respectively. The INEPT spectrum was acquired with 512 transients and 128 scans per transient. For the CP spectrum, the PARIS pulse sequence was used with 100 ms mixing time and 400 transients with 268 scans per transient. (b) Zoom into a region centered around the Arg Cβ–Cα crosspeaks. (c) Comparison of ChiZ1-64 and ChiZ-FL INEPT spectra, shown in black and red, respectively. (d) Zoom into the region centered around the Arg Cβ–Cα crosspeaks. All experiments were carried out at 25 °C and at a 12.2 kHz spinning rate.
Figure 4
Figure 4
Paramagnetic relaxation enhancement data of ChiZ1-64 bound to and ChiZ-FL reconstituted into POPG:POPE liposomes. The protein to lipid ratios were 1:50 and 1:80, and Gd3+-chelated lipids were at 2 and 1%, respectively, for the two constructs. (a) Molecular structure of POPG, POPG, and PE-DTPA (Gd). One-dimensional 13C direct-excitation spectra of (b) ChiZ1-64 and (c) ChiZ-FL in the absence (black) and presence (red) of Gd3+-chelated lipids. 128 scans were collected on each sample. (d) 1H–13C INEPT-based spectra of ChiZ-FL in the absence (black) and presence (red) of Gd3+-chelated lipids. The aliphatic and α-carbon regions are shown in two panels. All experiments were carried out at 25 °C and at a 12.2 kHz spinning speed.
Figure 5
Figure 5
Membrane-contact probabilities of NT residues. (a) Contact status of individual residues in snapshots along a 1.9-μs molecular dynamics trajectory of ChiZ1-64. Green bars and blanks indicate that a residue either is or is not in contact with the membrane. (b) Membrane-contact probabilities of NT residues in the three constructs. The shaded bands represent standard deviations among the snapshots analyzed. The extreme N-terminal residues that show high membrane-contact probabilities in ChiZ1-86 and ChiZ-FL are from two MD trajectories where Met1 was started as nearly embedded in the headgroup region, mimicking in a small way potential membrane attachment of the N-terminal His-tag; Met1 eventually dissociated from the membrane. For these two constructs, residues 49–56 penetrated into the membrane in two trajectories. These events led to relatively large standard deviations in membrane-contact probability. (c) A snapshot of ChiZ1-64 at 1.56 μs from the same trajectory as in (a), illustrating the membrane anchoring of NT by Arg residues in the midsection.
Figure 6
Figure 6
Hydrogen bonding probabilities of NT Arg residues. (a) Hydrogen bonding probabilities of Arg residues with POPG lipids. (b) Hydrogen bonding probabilities of Arg residues with Asp and Glu residues. (c) Hydrogen bonding probabilities of Arg residues with POPE lipids, scaled up by a factor of 7/3. (d) Average number of Arg residues that hydrogen bond with a particular type of partner at a given moment, and the counterpart for the partner hydrogen bonding with Arg residues. The partners are either POPG or POPE lipids or Asp and Glu residues.
Figure 7
Figure 7
Networks of membrane-contacting residues. (a–c) Contact networks of the three ChiZ constructs. Node radii are proportional to contact probabilities Ci; only nodes with Ci > 0.25 are shown. Edge widths are proportional to co-occurrence probabilities Cij; only edges with Cij > 0.20 are shown. (d) A snapshot of ChiZ-FL, illustrating residues that contact the membrane at the same time..
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wu S.; Wang D.; Liu J.; Feng Y.; Weng J.; Li Y.; Gao X.; Liu J.; Wang W. The Dynamic multisite interactions between two intrinsically disordered proteins. Angew. Chem., Int. Ed. 2017, 56 (26), 7515–7519. 10.1002/anie.201701883. - DOI - PubMed
    1. Borgia A.; Borgia M. B.; Bugge K.; Kissling V. M.; Heidarsson P. O.; Fernandes C. B.; Sottini A.; Soranno A.; Buholzer K. J.; Nettels D.; Kragelund B. B.; Best R. B.; Schuler B. Extreme disorder in an ultrahigh-affinity protein complex. Nature 2018, 555 (7694), 61–66. 10.1038/nature25762. - DOI - PMC - PubMed
    1. Turner A. L.; Watson M.; Wilkins O. G.; Cato L.; Travers A.; Thomas J. O.; Stott K. Highly disordered histone H1-DNA model complexes and their condensates. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (47), 11964–11969. 10.1073/pnas.1805943115. - DOI - PMC - PubMed
    1. Neira J. L.; Palomino-Schätzlein M.; Ricci C.; Ortore M. G.; Rizzuti B.; Iovanna J. L. Dynamics of the intrinsically disordered protein NUPR1 in isolation and in its fuzzy complexes with DNA and prothymosin α. Biochim. Biophys. Acta, Proteins Proteomics 2019, 1867 (11), 140252.10.1016/j.bbapap.2019.07.005. - DOI - PubMed
    1. Meyer N. H.; Dellago H.; Tam-Amersdorfer C.; Merle D. A.; Parlato R.; Gesslbauer B.; Almer J.; Gschwandtner M.; Leon A.; Franzmann T. M.; Grillari J.; Kungl A. J.; Zangger K.; Falsone S. F. Structural fuzziness of the RNA-organizing protein SERF determines a toxic gain-of-interaction. J. Mol. Biol. 2020, 432 (4), 930–951. 10.1016/j.jmb.2019.11.014. - DOI - PubMed