Direct Evidence for Deprotonation of a Lysine Side Chain Buried in the Hydrophobic Core of a Protein

Takayama, Yoshihisa; Castañeda, Carlos; Chimenti, Michael S.; García-Moreno, Bertrand; Iwahara, Junji

doi:10.1021/ja801731g

Cited by 54 publications

(113 citation statements)

References 15 publications

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“…In HIV-2 MA, Glu12 is substituted by Lys. Previous studies have shown that the p K a value of a buried Lys residue can be abnormally low (∼5.6) compared to Lys residues that are usually found at the surface and interact extensively with water (p K a = ∼10.5) (87). In this case, Lys side chain is present in the neutral state (NH 2 ).…”

Section: Discussionmentioning

confidence: 99%

Myristate Exposure in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Modulated by pH

et al. 2010

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Human immunodeficiency virus type-1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix (myr(+)MA) domain of Gag and phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P 2 ). Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P 2 binding and is enhanced by factors that promote protein self-association. In the studies reported herein, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His-Glu salt bridge by single amino acid substitutions greatly altered the myr-sequestered-myr-exposed equilibrium. In vivo intracellular localization data revealed that H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a "pH sensor" in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation. KeywordsHuman immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2); myristyl (myr); matrix (MA); nuclear magnetic resonance (NMR); His-Glu salt bridge Corresponding Author: Jamil S. Saad, Ph.D., 845 19 th Street South, Birmingham, AL 35294;; saad@uab.edu. SUPPORTING INFORMATION AVAILABLE 2D 1 H-15 N HSQC NMR spectra of myr(−) and myr(+)MA as a function of pH (Figs. S1 and S2); 3D 13 C-edited/ 12 C-double-halffiltered NOE spectrum of myr(+)MA at pH 5.5 (Fig. S3); sedimentation equilibrium profiles for myr(+)MA at different pH values (Fig. S4); plots of chemical shift changes vs. pH used to determine the pK a value of His89 for myr(−)MA (Fig. S5); sedimentation equilibrium and velocity profiles for WT CA and various MACA mutants as a function of pH ( Fig. S6-S9); 2D NMR HSQC spectra (Fig. S10), sedimentation velocity profiles ( Fig. S11) and CD spectra (Fig. S12) for MA H89G mutants; 2D HSQC spectra and sedimentation velocity profiles of MA E12A mutants (Fig. S13); sedimentation velocity profiles for myr(+)MA-E12A/W184A/ M185A as a function of pH (Fig. S14); structural representation of salt bridge formation in the HIV-1 MA structures (Fig. S15); and, SDS-PAGE showing the effect of His89 and E12 mutations on virus production (Fig. S16). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 20...

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Section: Discussionmentioning

confidence: 99%

Myristate Exposure in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Modulated by pH

et al. 2010

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“…Here, through the combination of long-range 15 NÀ 13 C scalar J-coupling measurements and an atomic-detail molecular dynamics (MD) simulation, direct insight into the structural dynamic behavior of lysine side chains in human ubiquitin has been gained. On the basis of 1 H/ 13 C/ 15 N heteronuclear correlation experiments selective for lysine NH 3 þ groups, we analyzed two different types of long-range 15 NÀ 13 C J-coupling constants: one between intraresidue 15 Nζ and 13 Cγ nuclei ( 3 J NζCγ ) and the other between 15 Nζ and carbonyl 13 C 0 nuclei across a hydrogen bond ( h3 J NζC 0 ). The experimental 3 J NζCγ data confirm the highly mobile nature of the χ 4 torsion angles of lysine side chains seen in the MD simulation.…”

Section: B S Supporting Informationmentioning

confidence: 99%

Signature of Mobile Hydrogen Bonding of Lysine Side Chains from Long-Range ¹⁵N–¹³C Scalar J-Couplings and Computation

Zandarashvili

Wang

et al. 2011

J. Am. Chem. Soc.

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Amino acid side chains involved in hydrogen bonds and electrostatic interactions are crucial for protein function. However, detailed investigations of such side chains in solution are rare. Here, through the combination of long-range (15)N-(13)C scalar J-coupling measurements and an atomic-detail molecular dynamics (MD) simulation, direct insight into the structural dynamic behavior of lysine side chains in human ubiquitin has been gained. On the basis of (1)H/(13)C/(15)N heteronuclear correlation experiments selective for lysine NH(3)(+) groups, we analyzed two different types of long-range (15)N-(13)C J-coupling constants: one between intraresidue (15)Nζ and (13)Cγ nuclei ((3)J(NζCγ)) and the other between (15)Nζ and carbonyl (13)C' nuclei across a hydrogen bond ((h3)J(NζC')). The experimental (3)J(NζCγ) data confirm the highly mobile nature of the χ(4) torsion angles of lysine side chains seen in the MD simulation. The NH(3)(+) groups of Lys29 and Lys33 exhibit measurable (h3)J(NζC') couplings arising from hydrogen bonds with backbone carbonyl groups of Glu16 and Thr14, respectively. When interpreted together with the (3)J(NζCγ)-coupling constants and NMR-relaxation-derived S(2) order parameters of the NH(3)(+) groups, they strongly suggest that hydrogen bonds involving NH(3)(+) groups are of a transient and highly dynamic nature, in remarkably good agreement with the MD simulation results.

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“…In other cases, the presence of polar or charged residues in unfavorable, hydrophobic regions can have detrimental consequences for protein stability and/or function 3,4 . Furthermore, many of the permissible mutations which place an ionizable residue in the hydrophobic core of a protein are accompanied by pK a shifts which render side chain non-charged 5,6 . In similar fashion, burying a polar amino acid in the hydrophobic interior of a lipid bilayer membrane can be challenging.…”

Section: Introductionmentioning

confidence: 99%

Accommodation of a Central Arginine in a Transmembrane Peptide by Changing the Placement of Anchor Residues

et al. 2012

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Both Trp and Arg in transmembrane protein domains make important interactions with lipids at the membrane/water interface, but at different depths. Derivatives of the designed peptide GWALP23, acetyl-GGALW5LALALALALALALW19LAGA-amide, with single Trp anchors, have proven useful for characterizing such interactions. Indeed previous work revealed quite different effects emanating from Arg substitutions at positions 12 and 14 within GWALP23, with the R12 peptide exhibiting multiple positions and orientations with respect to DOPC bilayer membranes (Vostrikov et al. [2010] J. Am. Chem. Soc. 132, 5803–5811). To gain further understanding of the multi-state behavior, we moved the Trp “anchor” residues to more outer positions 3 and 21 in GWALP23 itself, and in the R12 and R14 derivatives. The locations and orientations of the peptides with respect to lipid bilayer membranes of differing thickness were investigated by means of solid-state 2H NMR spectroscopy, using labeled alanines, and coarse-grained molecular dynamics simulations. Interestingly, relatively intense and narrow 2H resonances from selected backbone Cα deuterons were observed over quite narrow ranges of frequency and sample orientation. The backbone resonances reflect dynamic complexities and at the same time provide important contributions for the analysis of peptide transmembrane orientation. With the Trp3,21 anchors relatively far from the peptide and bilayer center, the results indicate significantly large apparent tilt angles, for example close to 30° for the new R12 and R14 peptides with respect to the bilayer normal of DLPC membranes. The R12 side chain indeed is “rescued” to a stable position, where it is accommodated within the transmembrane helix, when the Trp anchors are moved outward and to another face of the helix. At the same time, the R14 side chain of transmembrane GW3,21ALP23 also retains a stable favored position.

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Direct Evidence for Deprotonation of a Lysine Side Chain Buried in the Hydrophobic Core of a Protein

Cited by 54 publications

References 15 publications

Myristate Exposure in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Modulated by pH

Myristate Exposure in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Modulated by pH

Signature of Mobile Hydrogen Bonding of Lysine Side Chains from Long-Range ¹⁵N–¹³C Scalar J-Couplings and Computation

Accommodation of a Central Arginine in a Transmembrane Peptide by Changing the Placement of Anchor Residues

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Direct Evidence for Deprotonation of a Lysine Side Chain Buried in the Hydrophobic Core of a Protein

Cited by 54 publications

References 15 publications

Myristate Exposure in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Modulated by pH

Myristate Exposure in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Modulated by pH

Signature of Mobile Hydrogen Bonding of Lysine Side Chains from Long-Range 15N–13C Scalar J-Couplings and Computation

Accommodation of a Central Arginine in a Transmembrane Peptide by Changing the Placement of Anchor Residues

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Signature of Mobile Hydrogen Bonding of Lysine Side Chains from Long-Range ¹⁵N–¹³C Scalar J-Couplings and Computation