SASDAG7: CD44 HABD scFv MEM-85 complex (Hyaluronate binding domain of CD44...

Basic information

• Entry: Database: SASBDB / ID: SASDAG7

Sample

CD44 HABD scFv MEM-85 complex

• Hyaluronate binding domain of CD44 antigen (protein), CD44 HABD, Homo sapiens
• Single-chain Variable Fragment of Antibody MEM-85 (protein), scFv MEM-85, Mus musculus

• Biological species: Homo sapiens (human); Mus musculus (house mouse)
• Citation: Journal: J Struct Biol / Year: 2015; Title: Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85.; Authors: Jana Škerlová / Vlastimil Král / Michael Kachala / Milan Fábry / Ladislav Bumba / Dmitri I Svergun / Zdeněk Tošner / Václav Veverka / Pavlína Řezáčová / ; Abstract: The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD-antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD-scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.

Contact author

• Michael Kachala (EMBL-Hamburg, European Molecular Biology Laboratory (EMBL) - Hamburg outstation, Notkestraße 85, Geb. 25A, 22607 Hamburg, Deutschland, Germany)

Models

• Model #279: ; Type: dummy / Software: DAMMIN / Radius of dummy atoms: 2.50 A / Symmetry: P1 / Chi-square value: 2.460; Search similar-shape structures of this assembly by Omokage search (details)
• Model #280: ; Type: atomic / Software: SASREF / Radius of dummy atoms: 1.90 A / Chi-square value: 1.24; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: CD44 HABD scFv MEM-85 complex / Specimen concentration: 1.30-5.00 / Entity id: 158 / 159
• Buffer: Name: PBS / PK: 7 / pH: 7.4

Entity #158

Name: CD44 HABD / Type: protein / Description: Hyaluronate binding domain of CD44 antigen / Formula weight: 17.9 / Num. of mol.: 1 / Source: Homo sapiens
Sequence:

SNAASQIDLN ITCRFAGVFH VEKNGRYSIS RTEAADLCKA FNSTLPTMAQ MEKALSIGFE TCRYGFIEGH VVIPRIHPNS ICAANNTGVY ILTSNTSQYD TYCFNASAPP EEDCTSVTDL PNAFDGPITI TIVNRDGTRY VQKGEYRTNP EDIYPSNPTD DDV

Entity #159

Name: scFv MEM-85 / Type: protein
Description: Single-chain Variable Fragment of Antibody MEM-85
Formula weight: 28.5 / Num. of mol.: 1 / Source: Mus musculus
Sequence:

EVQLQESGPG LVAPSQSLSI TCTVSGFSLT NYGVHWVRQP PGKGLEWLGV IWAGGSTNYN SALMSRLSIS KDNSKSQVFL KMNSLQTDDT AMYYCARDGA RAMDYWGQGT TVTVSGGGGS GGGGSGGGGS GGGGSDIVMS QSPSSLAVSV GEKVTVSCKS SQSLLYSSNQ KNYLAWYQQK PGQSPKLLIS WASTRESGVP DRFTGSGSGT DFTLTISSVK AEDLAVYYCQ QSYSYPWTFG GGTKLEIKRE QKLISEEDLN GTHHHHH

Experimental information

• Beam: Instrument name: PETRA III P12 / City: Hamburg / 国: Germany / Type of source: X-ray synchrotron / Wavelength: 0.12 Å / Dist. spec. to detc.: 3.1 mm
• Detector: Name: Pilatus 2M

Scan

Title: CD44 HABD scFv MEM-85 complex / Measurement date: Oct 31, 2013 / Storage temperature: 10 °C / Cell temperature: 10 °C / Exposure time: 0.05 sec. / Number of frames: 20 / Unit: 1/nm /

	Min	Max
Q	0.2003	4.4884

Distance distribution function P(R)

Sofotware P(R): GNOM 4.5a / Number of points: 352 /

	Min	Max
Q	0.2029	2.973
P(R) point	1	352
R	0	9.415

Result

Type of curve: extrapolated / Standard: BSA /

	Experimental	Porod	Porod error
MW	44 kDa	-	-
Volume	-	57 nm3	200

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	8363	135	8235	83
Radius of gyration, Rg	2.81 nm	0.05	2.69 nm	0.14

	Max	Error
D	9.4	1