NMR solution structure of the 32-kDa platelet factor 4 ELR-motif N-terminal chimera: a symmetric tetramer

Mayo, Kevin H.; Roongta, Vikram; Ilyina, Elena; Milius, Robert P.; Barker, Sharon; Quinlan, Christopher; Rosa, Greg La; Daly, Thomas J.

doi:10.1021/bi00036a012

Cited by 61 publications

(83 citation statements)

References 59 publications

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“…fragments, the 5Ј sequences of which correspond to the C termini of the PF4 deleted fragments PF4 1-60 , PF4 , and PF4 1-40 , PF4 , PF4 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] , PF4 [1][2][3][4][5][6][7][8][9][10][11][12][13] , PF4 ⌬41-50 , and PF4 ⌬45-48 (Fig. 1B).…”

Section: Methodsmentioning

confidence: 99%

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Evidence for a Granule Targeting Sequence within Platelet Factor 4

Issertial

Rosa²,

Briquet-Laugier³

2005

Journal of Biological Chemistry

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Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed ␣-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory ␣-granule chemokine, and demonstrated that PF4 ␣-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 ؎ 2% to 17 ؎ 3%, p < 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in ␣-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNGgreen fluorescent protein chimera exhibited an 82.5 ؎ 1.8% colocalization with the ␣-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human ␣-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same ␣-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to ␣-granule targeting of chemokines and possibly of other ␣-granule proteins.

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Section: Methodsmentioning

confidence: 99%

“…PF4 is an antiangiogenic factor that inhibits megakaryocyte and endothelial cell proliferation (15,16). Importantly, the PF4 structure has been solved by x-ray cristallography, allowing structure-function predictions (17,18). PF4 is released along with other ␣-granule proteins upon physiological and pathological stimulation (9).…”

mentioning

confidence: 99%

Evidence for a Granule Targeting Sequence within Platelet Factor 4

Issertial

Rosa²,

Briquet-Laugier³

2005

Journal of Biological Chemistry

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“…PF4M2 is a PF4 chimera in which the N terminus of PF4 is replaced with the N terminus of IL8. Because PF4M2 forms symmetric tetramers resulting in a well defined set of cross-peaks, complete 1 H/ 15 N resonance assignments and structure analysis were possible (7). By comparing 1 H-15 N HSQC spectra for PF4M2 and native PF4, some tentatively assigned cross-peaks for native PF4 have been labeled in the Fig.…”

Section: [D⅐r] Is the Molar Concentration Of Il8 Dimer (D)-receptor (mentioning

confidence: 99%

“…PF4M2 functions biologically like native PF4 (7). However, PF4M2 forms symmetric tetramers, greatly simplifying its NMR spectra and allowing nearly complete 1 H/ 15 N resonance assignments to be made and the structure to be determined (7). Performing the same IL8 titration study using 15 N-labeled PF4M2 has allowed more detailed insight into how PF4 and IL8 interact.…”

Section: Interactions Between Pf4 (Pf4m2) and Il8 Defined Using Nmr-tomentioning

confidence: 99%

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Platelet Factor 4 and Interleukin-8 CXC Chemokine Heterodimer Formation Modulates Function at the Quaternary Structural Level

Nesmelova

Sham

Dudek

et al. 2005

Journal of Biological Chemistry

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The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel ␤-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.Platelet factor-4 (PF4) 1 and interleukin-8 (IL8) are members of the CXC chemokine family of small (8 -10 kDa) proteins, a subfamily of chemokines within the cytokine superfamily (1). CXC chemokines are generally chemotactic for migratory immune cells, and thus are involved in the regulation of inflammatory processes and wound healing (1-4). In addition, they demonstrate biological activities in hematopoiesis, cell proliferation, angiogenesis, and glycosaminoglycan binding (1-4).CXC chemokines, which are known to self-associate as dimers and tetramers, exhibit high sequence and three-dimensional structural homology at both the tertiary (monomer) and quaternary (dimer and tetramer) levels. Each folded CXC chemokine monomer has an aperiodic, two-disulfide bond-stabilized N-terminal segment, followed by a three-stranded antiparallel ␤-sheet domain, and a C-terminal ␣-helix that is folded onto the generally amphipathic ␤-sheet (5). Dimers are formed by intermolecularly extending the monomer ␤-sheet into a sixstranded antiparallel ␤-sheet (originally termed AB-type dimer (6)). Tetramers are formed by ␤-sheet sandwiching of two ABtype dimers (6). Native PF4 dimers associate asymmetrically into tetramers (6), whereas in an N-terminal PF4 chimera (PF4M2), dimers associate symmetrically into tetramers with little change in biological activity (7). IL8 forms AB-type dimers and is not known to tetramerize (8 -10).Despite sequence and structure similarities, PF4 and IL8 mediate different biological activities. Although first recognized to bind heparin and act as an anticoagulant, PF4 is also known as an anti-angiogenic agent (11). On the other hand, IL8 binds heparin more weakly and can promote angiogenesis (11,12). The angiogenic functional difference may be due, at least in part, to N-terminal sequence differences. PF4 lacks the IL8 N-terminal tripeptide Glu-Leu-Arg (ELR) motif, which is known to mediate IL8 receptor binding and subsequent signal transduction (13). In additi...

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A recipe for designing water‐soluble, β‐sheet‐forming peptides

1996

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Based on observations of solubility and folding properties of peptide 33-mers derived from the &sheet domains of platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-a), as well as other 6-sheet-forming peptides, general guidelines have been developed to aid in the design of water soluble, self-association-induced 0-sheet-forming peptides. CD, 'H-NMR, and pulsed field gradient NMR self-diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native-like @-sheet tetramers and is sparingly soluble above pH 6. IL-8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, nativelike &sheet dimers at higher pH. Gro-a peptide is soluble at all pH values, yet displays no discernable @-sheet structure even when diffusion data indicate dimer-tetramer aggregation. A recipe used in the de novo design of water-soluble 0-sheet-forming peptides calls for the peptide to contain 40-50~0 hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noncharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33-residue peptides are presented supporting this approach; Under near physiologic conditions, all four peptides are soluble, form &sheet structures to varying degrees, and self-associate. One peptide folds as a stable, compact 0-sheet tetramer, whereas the others are transient 6-sheet-containing aggregates.

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NMR solution structure of the 32-kDa platelet factor 4 ELR-motif N-terminal chimera: a symmetric tetramer

Cited by 61 publications

References 59 publications

Evidence for a Granule Targeting Sequence within Platelet Factor 4

Evidence for a Granule Targeting Sequence within Platelet Factor 4

Platelet Factor 4 and Interleukin-8 CXC Chemokine Heterodimer Formation Modulates Function at the Quaternary Structural Level

A recipe for designing water‐soluble, β‐sheet‐forming peptides

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