The Human Complement System

1
The Human Complement System
Components

• 35 different blood proteins
• Enzymes
• Receptors
• Regulatory proteins

Functions

• Killing of pathogenic microorganisms (MAC)
• Clearance of immune complexes
• Acute inflammatory reactions

2
Assembly of the Membrane Attack Complex (MAC)
Lectin Pathway
3
The MAC Pore
5 - 10 nm
4
Assembly of the Membrane Attack Complex (MAC)
Lectin Pathway
5
Subunits of Human C8
C8g (22 kDa)
N
C
LIPOCALIN
S
S
T1
LA
MACPF
EG
T1
C
C8a (64 kDa)
N
?
? ? ? ? ?
? ? ? ? ?
? ? ? ? ?
? ? ? ? ?
T1
C8b (64 kDa)
LA
MACPF
EG
T1
N
C
6
Binding Sites on C8
C8g
N
C
LIPOCALIN
S
S
T1
LA
MACPF
EG
T1
C
C8a
N
C8g
CD59
C8b
C9
C8b
T1
LA
MACPF
EG
T1
N
C
C8a
C5b-7
7
Crystallization of Human C8g and Data Collection
Recombinant C8g
40 PEG 4000, 0.1M Na Citrate, pH 4
Vapor Diffusion Hanging Drop Method
Single Crystals 0.2 x 0.15 x 0.05 mm
Mother Liquor 0.95 M KBr
Xe at 230 psi
MAD
SAD
40 PEG 4000, 0.1 M Na Citrate, 1mM K2PtCl4 pH 7
MAD
Xe Derivatized Crystals
Br- Derivatized Crystals
Pt Derivatized Crystals
8
Electron Density Map at 1.2 Å Resolution
Phe
Trp
Pro
9
3D Structure of C8?
10
Citrate Ion Modeled into Maps of Electron Density
and Surface Charge
Positive charge in blue Negative charge in red
11
Cross-sectional View of the C8g Ligand Binding
Site
Hydrophilic upper portion
Xe in the Lower Cavity
Y131
Y83
Hydrophobic lower cavity
12
C8g-Lauric Acid (C120) Complex
13
Superposition of the C8g, C8g-Xe and C8g-Laurate
Structures
Native C8g (yellow) Xe (red) Lauric Acid (blue)
14
Activity of C8g
Relative Activity ()
Hemolytic Bacterial Killing
C8?-? C8b ? C8 100
100 C8? C8? ? C8? 10 80
C8? C8? C8? ? C8 60 95
15
Summary

• C8? DISPLAYS A TYPICAL LIPOCALIN FOLD
• THE CALYX ENTRANCE IS () CHARGED
• MOVEMENT OF Y83 AND Y131 ALLOWS ACCESS TO A LOWER
  HYDROPHOBIC CAVITY
• LIGAND IS (-) CHARGED AT ONE END, HYDROPHOBIC AT
  THE OTHER
• POSSIBLE FUNCTIONS FOR C8?
• BIND MEMBRANE LIPID SIDE CHAINS
• BIND A SMALL LIPID-LIKE MOLECULE PRODUCED IN
  THE VICINITY OF THE MAC