Transcript ICAM-1: Host and Viral Receptors

Intercellular Adhesion Molecule-1:
binding to cellular and viral ligands, with
implications for biotherapeutics
Jin Lab Retreat, August ‘08
Róisín Owens
Use of ICAM-1 for biotherapeutics:
1. Binding to LFA-1 I domain
Why? - Important for inhibiting certain immune responses (e.g preventing
immune rejection in organ transplantation). Crystallization of complexes
helps to design other small molecule inhibitors alongside D1
Aim: - Characterization and crystallization of LFA-1 I domain/ ICAM-1 D1
complex
2. Binding to Rhinovirus
Why? - Important as an antiviral
Aim: - Identification and Characterization of D1 mutants capable of potently
inhibiting Rhinovirus, and crystallization of Rhinovirus/ ICAM-1 D1 complex
ICAM-1
ICAM-1 serves as a counter
receptor for leukocyte integrins
•LFA-1 (lymphocyte function-associated
antigen-1; L2)
•Mac-1 (M2)
ICAM-1 is also receptor for HRV
D1 LFA-1/ HRV
D2
D3 Mac-1
Glycosylation
D4
D5
Aims (1)
Characterization of the binding of ICAM-1 D1
variants to HRV serotypes to identify mutants that
are highly potent in binding and inhibiting cell
infection
•SPR analysis of HRV binding to ICAM-1 D1 variants
•Analysis of effect of D1 binding to viral capsids
•Measurement of cytopathic effects of D1 variants
•Structure of D1 in complex with HRV
Research Design and Methods (1)
1.1 SPR analysis of HRV binding to ICAM-1 D1 variants
•Biotinylate D1 variants, immobilize on chip
•Immobilize virus
•Measure affinity of different mutants to different HRV serotypes
1.2 Analysis of effect of D1 binding to viral capsids
•Mix D1 with virus in different concentrations and assess sedimentation in a sucrose
gradient
•Radiolabel/ Fluorescently label virus to track
1.3 Measurement of cytopathic effects of D1 variants
•Use Epic system to assess effect of D1 mutants in inhibiting viral infection
1.4 Structure of D1 in complex with HRV
•Based on previous tasks, identify best complex of HRV serotype and D1 mutant for
crystal structure
Preliminary Data (1.1)
Binding of HRV16 to ICAM-1 Fc by SPR
80
70
4nM
5nM
60
50
3nM
RU
40
Binding of HRV16
2nM
30
20
1nM
10
0
0
50
100
150
20060
250
300
0nM350
-10
-20
Time (s)
10X
50
3 nM
1X
Inhibition of
binding of HRV16
by ICAM-1 D1.v3
RU
40
30
1000X
20
10
0
0
-10
50
100
150
200
Time (s)
250
300
0 nM
350
Preliminary Data (1.2)
Purification of HRV16
Absorbance at 495nm
3.5
3
Ro s s ma nn
2.5
ELISA of purified
HRV16;
comparison of
two preps
Ro is in
2
1.5
1
0.5
0
1000ng
SDS-PAGE
of purified
HRV16
500ng
100ng
50ng
10ng
5ng
TEM of
purified
HRV16
1ng
0ng
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
200nm
Preliminary Data (1.3)
Inhibition of Rhinovirus entry by ICAM-1 D1D5 quantitation of cell death with crystal violet
CPE assay
Absorbance at 570nm
Absorbance at 570nm
3.0000
3.5
2.5000
2.0000
1.5000
1.0000
0.5000
0.0000
4
3
19
23
27
Time (hours)
2.5
BSA
D1D5
2
1.5
1
0.5
0
0 µg
1 µg
0.5 µg
0.1 µg
0.05 µg
Protein Amount
0.01 µg
0.005 µg
43
50
2ug
200ng
20ng
2ng
200pg
20pg
2pg
200fg
20fg
2fg
200ag
20ag
no virus
Preliminary Data (1.4)
Rhinovirus infection of HeLa cells using the EPIC platform
Cell growth
On Epic plate
Cells seeded
15K/well
Virus infection
Continuous scan in the reader (26 C, no CO2)
Response (pm)
24 hr
Pfu/cell
11hr
Time (sec)
Changqing Wang & Arron S. Xu, Corning
17hr
22hr
Preliminary Data (1.5)
Rhinovirus infection of HeLa cells using the EPIC platform: Inhibition assay
Aims (2)
Characterization of selected LFA-1 I domain – ICAM-1 D1
binding partners with subsequent structure
determination
•SPR analysis of active conformation I domain mutants to
ICAM-1 D1 variants
•Structure of D1 in complex with active conformation I
domain
Research Design and Methods (2)
1.1 SPR analysis of I domain mutants binding to ICAM-1 D1
variants
•Biotinylate D1 variants, biotinylate I domains, immobilize on chip
•Measure affinity
1.4 Structure of D1 in complex with HRV
•Based on previous task, identify best complex of I domain and D1 mutants for crystal
structure
Preliminary Data (2.1)
Have generated an array of HA I domain mutants* with N-terminal Cys
for SPR measurements of I-domain in complex with ICAM-1 D1
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Ribbon diagram showing docking of the LFA-1 Idomain (green) with ICAM-1 D1 (orange)
Rossmann, PNAS 1998
F265S/F292G
K287C/K294C
F277L/F292A
F265S/F292A
F277L
F292A
*HA I domains mimic
open conformation of
LFA-1
Preliminary Data (2.2)
Binding of ICAM-1 D1.v3 to HA I-domain by SPR
140
120
100
RU
80
60
40
20
0
0
50
100
150
200
250
300
350
400
450
-20
Time (s)
Biotinylated F265S/F292G I domain, Immobilized on Streptavidin chip
Preliminary Data (2.3)
ICAM-1 D1/ HA I-domain complex Size exclusion FPLC
Elution of complex
800
Absorbance units (A280)
are needed to see this picture.
TIFF (U ncompressed) decompressor
QuickTime™ and a
I-domain
700
ICAM-1 D1
600
500
F277L/F292A
F265S/F292G
400
300
Free ICAM-1 D1
200
100
0
0
5
10
15
-100
Volume (ml)
20
25
30
Preliminary Data (2.4)
ICAM-1 D1/ HA I-domain complex Ion exchange FPLC
Absorbance units (A280)
30
25
I domain
ICAM-1 D1
20
Free I domain/
free ICAM1 D1
complex
15
10
5
0
0
-5
2
4
6
8
Volume (ml)
10
12
14
16
Preliminary Data (2.5)
ICAM-1 D1/ HA I-domain complex Crystallization
• Brought microcrystals to CHESS
• Diffracted to ~8 Å
• Hit was with I domain F265S/F292G