Crystallographic Insight into Collagen Recognition by Discoidin Domain Receptor 2

Carafoli, Federico; Bihan, Dominique; Stathopoulos, Stavros; Konitsiotis, Antonios D.; Kvansakul, Marc; Farndale, Richard W.; Leitinger, Birgit; Hohenester, Erhard

doi:10.1016/j.str.2009.10.012

Cited by 117 publications

(151 citation statements)

References 55 publications

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“…Sequence alignment demonstrated conservation of around 50% with DSDs in related proteins, CPX-2 and ACLP, and around 30% with DSDs from non-related proteins, with the highest identity shared with the collagen-binding DSDs from the DDRs. More detailed analysis revealed conservation of key residues involved in mediating the DDR-DSD -collagen interaction via the formation of an amphiphilic binding trench [16,17]. These residues are also conserved in CPX-2 and ACLP, with the latter reported to associate with collagen I in studies implicating ACLP in pulmonary fibrosis [18].…”

Section: Accepted Manuscriptmentioning

confidence: 94%

“…The molecular details of the DSD-collagen interaction have been elucidated through a combination of NMR studies [15], resolution of the crystal structure of the DDR2-DSD-collagen complex [16], and functional investigations exploring the collagen-binding site within the DDR1-DSD [17]. Together, these studies indicate that the four surface-exposed loops situated within the DSD combine to form an amphiphilic binding trench that binds to the GVMGFO motifs present in fibrillar collagens I-III [15,16,17]. Key residues that (Fig 2).…”

Section: In Silico Analysis Suggests the Dsd Of Cpx-1 May Bind Collagenmentioning

confidence: 99%

“…R192 residue situated within the third loop of the DSD (see Fig 2) and integral to the binding trench [16] was mutated to Ala. A previous report showed that mutation of the equivalent residue in DDR1 (R105) reduced collagen binding by 80% [17]. We compared the expression, secretion and binding characteristics of CPX-1 R192A and wild-type (WT) CPX-1.…”

Section: The Dsd Is Required For Cpx-1 Binding To Collagenmentioning

confidence: 99%

“…It is tempting to speculate that, like the DDRs, CPX-1, as well as CPX-2 and ACLP, binds to the GVMGFO motif common to the fibrillar collagens I-III [15,16,17]. Detailed investigations have established relative specificity with respect to DDR-binding to alternate, non-fibrillar collagens such as the collagens IV and X and have also revealed that such specificity is defined by nonconserved residues located at the periphery of the binding trench [26].…”

Section: Accepted Manuscriptmentioning

confidence: 99%

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Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Kim

O’Neill

Whitehead

2015

Biochemical and Biophysical Research Communications

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Carboxypeptidase X-1 (CPX-1) is an atypical member of the carboxypeptidase (CP) family of proteins involved in a variety of physiological and pathological processes. However, unlike most other family members CPX-1 lacks catalytic activity making its biological function unclear. CPX-1 contains a 160 amino acid discoidin domain (DSD) that serves as a binding domain in other proteins prompting us to investigate a putative functional role for this domain in CPX-1.Sequence alignment confirmed the overarching homology between the DSD of CPX-1 and other DSDs whilst more detailed analysis revealed conservation of the residues known to form the collagen-binding trench within the DSD of the discoidin domain receptors (DDRs) 1 and 2.Biochemical characterisation of transiently expressed human CPX-1 revealed that CPX-1 was secreted in an N-glycosylation-dependent manner as treatment with the N-glycosylation inhibitor tunicamycin inhibited secretion concomitant with a reduction in CPX-1 mobility on Western blot. Using a collagen pull-down assay we found that secreted CPX-1 bound collagen and this appeared independent of N-glycosylation as treatment with PNGaseF did not affect binding. Further analysis under non-reducing and reducing (+DTT) conditions revealed that CPX-1 was secreted in both monomeric and dimeric forms and only the former bound collagen.Finally, mutation of a key residue situated within the putative collagen-binding trench within the DSD of CPX-1 (R192A) significantly reduced secretion and collagen-binding by 40% and 60%, respectively. Collectively these results demonstrate that CPX-1 is a secreted collagen-binding glycoprotein and provide a foundation for future studies investigating the function of CPX-1.

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Section: Accepted Manuscriptmentioning

confidence: 94%

Section: In Silico Analysis Suggests the Dsd Of Cpx-1 May Bind Collagenmentioning

confidence: 99%

Section: The Dsd Is Required For Cpx-1 Binding To Collagenmentioning

confidence: 99%

Section: Accepted Manuscriptmentioning

confidence: 99%

See 2 more Smart Citations

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Kim

O’Neill

Whitehead

2015

Biochemical and Biophysical Research Communications

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“…It has previously been suggested that stimulating DDR1 with its ligand, collagen, causes aggregation [32][33][34]. Here, we label DDR1 with a SNAP-tag that has been stochastically linked to Alexa488 or Alexa546.…”

Section: Resultsmentioning

confidence: 99%

Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

et al. 2017

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Abstract:We present a new microscope integrating super-resolved imaging using structured illumination microscopy (SIM) with wide-field optically sectioned fluorescence lifetime imaging (FLIM) to provide optical mapping of molecular function and its correlation with biological nanostructure below the conventional diffraction limit. We illustrate this SIM + FLIM capability to map FRET readouts applied to the aggregation of discoidin domain receptor 1 (DDR1) in Cos 7 cells following ligand stimulation and to the compaction of DNA during the cell cycle.

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Surface‐bound collagen 4 is significantly more stable than collagen 1

Stynes

Kiroff

Page

et al. 2017

J Biomedical Materials Res

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Collagen 1 (C1) is commonly used to improve biological responses to implant surfaces. Here, the stability of C1 was compared with collagen 4 (C4) on a mixed macrodiol polyurethane, both adsorbed and covalently bound via acetaldehyde glow discharge polymerization and reductive amination. Substrate specimens were incubated in solutions of C1 and C4. The strength of conjugation was tested by incubation in 8 M urea followed by enzyme linked immunosorbent assays to measure residual C1 and C4. The basal lamina protein, laminin-332 (L332) was superimposed via adsorption on C4-treated specimens. Keratinocytes were grown on untreated, C1-treated, C4-treated, and C4 + L332-treated specimens, followed by measurement of cell area, proliferation, and focal adhesion density. Adsorbed C4 was shown to be significantly more stable than C1 and covalent conjugation conferred even greater stability, with no degradation of C4 over twenty days in 8 M urea. Cell growth was similar for C1 and C4, with no additional benefit conferred by superimposition of L332. The greater resistance of C4 to degradation may be consequent to cysteine residues and disulphide bonds in its non-collagenous domains. The use of C4 on implants, rather than C1, may improve their long-term stability in tissues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1364-1373, 2017.

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Crystallographic Insight into Collagen Recognition by Discoidin Domain Receptor 2

Cited by 117 publications

References 55 publications

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

Surface‐bound collagen 4 is significantly more stable than collagen 1

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