Quaternary Structure of the Collagen IV a1256 Hexamer

1
Quaternary Structure of the Collagen IV a1256
Hexamer

• Wes Robertson
• Mentors Dr. Billy Hudson, Dr. Roberto Vanacore
• BSCI 296

2
Background
OUTLINE -Collagen IV is an ancient,
highly-conserved protein that is one of the
primary structural components of the BM -BM is a
layer of ECM underlying the epithelial cells of
tissues, where they and one of their primary
functions is to provide structural support to
tissues -we know collagen IV is significant due
to diseases affecting col4, which lead to
impaired function of the BM. -For example, GD
autoimmune disease, AS genetic disease -to
understand function of collagen iv, we should
understand the structure of collagen IV

• Collagen IV Diseases
• Goodpastures Disease
• Alports Syndrome
• Diabetes

Epithelial cells
Collagen IV
3
Glomerulus? BM-col4-aorta mechanical stability to
tissues-col4 and BM required for necessary
function-if notDISEASES!
Collagen IV
OUTLINE -6 isoforms, which combine to make
triple-helical protomers -Only 3 known types of
protomers 121, 345, 565(putative!) -3 domains of
protomers 7S, collagenous, NC1noncollagenous. -N
C1 terminates as an NC1 trimer, which are
important because they combine with other NC1
trimers to form NC1 hexamers, thus leading to the
formation of supramolecular networks (below
laser) -networking ability confers structural
stability to BMs -there is a selectivity for Nc1
hexamer formation 121 hex, and 345 hex.,
suggested that the putative
the six chains of collagen IV apparently form
only three sets of triple helical molecules
called protomers, which are designated as 121,
345, and 565. These protomers
create collagenous networks by uniting two NC1
trimers to form hexamers and uniting four 7S
domains to form tetramers with other protomers,
how to get back to the 565 trimer without
explaining the 1256 network yet!
4
OUTLINE -121 hexamer structure known bond is
present -345 hex structure known bond is
present -1256 only suggested structure , where
previous research with chain-specific mAbs
determined the composition, but didnt provide
covalent linkages between monomers -bond has yet
to be investigated in the putative 1256 network
a121
a345
?
a1256
5
Hypothesis
a1-a5 and a2-a6 NC1 peptides are crosslinked by
the sulfilimine bond
Put bonds here?
6
Experimental Strategy
OUTLINE -look at aa sequences (?)
OUTLINE -SN covalent linkage between Met and
Hyl/Lys of NC1 domains from two trimers-therefore
reinforcing hexamers
Sulfilimine Bond
7
Collagen IV Sample Preparation
-In order for MS analyses, we must isolate
collagen IV hexamers -we chose bovine aorta, a
SMC-containing tissue, in which the Medial layer
BM contains both the 121 and 1256 col4
networks -provides a source of nc1 hexamers
Bovine Aorta
8
OUTLINE -With the pool of NC1 hexamers isolated
from bovine aorta BM. -collagenase enzyme digests
the middle collagenous domain leaving 7s and
NC1 -size-exclusion chromatography -hexamers
denature to dimer and monomeric subunits -WB with
chain-specific mAbs shows that 1256 chains
present any combination of 121 and 1256 hexamers
S300 Isolation of Bovine Aorta NC1 Hexamers
NC1
mAU 280nm
7S
Volume (ml)
9
Notes to isolate the a1256 NC1 hexamer, MonoS
anion exchange chromatography was used to
fractionate aorta NC1 hexamers
MonoS Isolation of a1256 NC1 Hexamer
Pk2 label with 1256 hexamer. Now know the
composition via chain-specific mAbs
Notes Bovine aorta NC1 hexamers were loaded into
the Mono S column and eluted with increasing
concentrations of NaCl absorbance was monitored
at 280 nm. -Western blot analysis of each MonoS
peak showed the a5 and a6 NC1 subunits eluting
most abundantly in peak 2, with the a1 and a2 NC1
subunits also found in this peak
3
3

2
Anti-a2
Anti-a1
mAU 280nm
mM NaCl
1
3
3
Anti-a5
Anti-a6
Volume (ml)
Peak 2 a1256 NC1 hexamer Peak 3 a121 NC1
hexamer
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Peptide Total Mass 4 m/z 5 m/z   6 m/z   7 m/z  
a1-a1 5010.4718 1253.62 1003.10 836.08 716.79
a2-a2 6163.0749 1541.78 1233.62 1028.19 881.45
a2-a6 5290.6067 1323.66 1059.10 882.78 756.80
a1-a5 3941.8956 986.4818 789.387 657.9905 564.1343
OUTLINE -MonoS peak 2 was digested with trypsin,
which cleaves the NC1 hexamers into smaller
peptides -know the aa sequence, so I did an in
silico trypsin digest to produce theoretical
peptide masses combining two with hyl and met
(-2 amu) -Table shows theoretical peptide masses
-2 amu to account for bond formationimportant
beause masses can be searched for in the mass
spectrometry analysis
Trypsin Digestion
11
Fractionation of Tryptic Peptides from a1256 NC1
Hexamer
-lyophilized and sent to mass spectrometry core
at VanderbiltVelos
mAU 214nm
3000-6000 Da
Volume (ml)
Predicted M/Z of MS1 ions from the bovine a1256
hexamer
Peptide Total Mass 4 m/z 5 m/z 6 m/z 7 m/z
a6- a2 6105.0696 1527.28 1222.02 1018.50 873.16
a2- a6 5290.6067 1323.66 1059.10 882.78 756.80
a1- a5 7777.5925 1945.40 1556.50 1297.30 1112.1
a5- a1 3925.894 982.50 786.20 656.30 561.85
12
Notes In order to detect sulfilimine-crosslinked
NC1 peptides, we used a high-resolution mass
spectrometry approach -Orbitrap Velos
high-resolution with higher scanning rate for
increased sensitivity in order to detect
sulfilimine-crosslinked peptides
Mass Spectrometry Analysis of Tryptic Peptides
Orbitrap Velos box???
Mass Spectrometry
Notes bioinformatics identify proteins in the
mass spectra by searching against the bovine
subset of the Uniref100 database.
Manual analysis
Bioinformatics
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Results
14
Full Scan of a5 Met93 crosslinked to a1 Hyl211
Notes loss of 2 amu in FS
100
90
80
70
60
Relative Abundance
50
T-3957
40
990.4786
30
z4
20
10
0
600
700
800
900
1000
1100
1200
1300
m/z
15
MS2 of m/z792.5850 (5)
730.7
z2
100
90
80
70
60
Relative Abundance
50
40
30
20
10
0
600
700
800
900
1000
1100
1200
1300
m/z
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Mass Spectrometry Results Summary
Sulfilimine-Crosslinked Peptide Mass
a6-a2 4393.2449
a2-a6 5290.6067
a5-a1 3941.8956
a1-a5 4943.3538
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3D Homology Model of a1256 NC1 Hexamer Quaternary
Structure
-RMS values 0.15 Angstroms -The best quality
models exhibit main-chain deviations no greater
than 0.4 Å,  -sequence identity A1-a5 191/229
(83.4) A2-a6 170/229 (74.2)
Notes A homology model of the a1256 NC1 hexamer
was constructed with SWISS-MODEL server and
visualized with UCSF Chimera
-Deposit to PDB -add more about atomic
coordinates, structure -2 steps
Notes showsthat the 121 trimer interacts with
the 565 trimer through a planar trimer-trimer
interface, comparable o the homotypic
interactions of 121 and 345 hexamers, yet
distinct in terms of it being heterotypic
(6/hex.) -565 trimer first chemical evidence of
its quaternary organization!
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Conclusions
OUTLINE -all collagen IV networks exhibit the
canonical hexameric quaternary structure at the
NC1 domain- -from this, we see that sulfilimine
bond is common to all six collagen IV isoforms
and common to all three collagen IV networks.
a121
a345
a1256
19
Acknowledgements

• Proteomics Laboratory
• Hayes McDonald, Ph.D
• Kristie Rose, Ph.D

• Billy G. Hudson, Ph.D
• Roberto Vanacore, Ph.D
• Kathy Friedman, Ph.D
• Suraj Adhikary
• Mohamed Rafi
• Parvin Todd
• Aaron Fidler
• Dorin-Bogdan Borza, Ph.D

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Cl- H2O2
HOCl
NC1 Hexamer
PXDN
21
-Collagen IV is an ancient, highly conversed
protein, which is a primary structural component
of basement membranes, a layer of ECM underlying
epithelial cells of all tissues -imparts
structural function by understanding the
structure of collagen IV 6 isoforms, which
combine to make triple-helical protomers -3 types
of protomers 121, 345, 565 -3 domains of
protomers NC1noncollagenous -protomers combine
at the C-termini NC1 domain to form NC1 hexamers,
allowing us to study collagen IV network
assembly -hexamers reinforced by the covalent
linkage, the SN bond conferring structural to
the BM in which the collagen IV networks reside
22
Alports Syndrome
Loss of the a1256 network from the aorta BM of
Alports patients has been correlated with an
increased susceptibility to aortic aneurysms
.Thus, COL4A5 mutations underscore a significant
yet unknown structural role of the a1256 network
for vascular tissue function.
-Alports syndrome is a genetic disease which
primarily results from nonsense mutations in the
COL4A5 gene. -In AS, COL4A5 mutations lead to
loss of the a5 and a6 chains in SMC BM (aorta,
bladder tissues)led to 565 trimer, as for the
hexamer -glean useful information from
becauseanalogous situation also in AS,
immunohistochemistry studies shown that COL4A5
mutations lead to loss of the a3-a5 chains in
GBM, which is explained by failed assembly and
concomitant absence of a345 network, which leads
to nephropathy and progresses to ESRD. -So, in
an analogous mechanism, loss of the 5 chain leads
to loss of the a6 chain due to incorporation into
same collagen IV network presumed a1256
network! -Borza provided explanation, where AS
leads to concomitant absence of the a1256
networks.
Absence of a345 network
nephropathy
Aortic abnormalities
???
Add trimer to top 345 and then keep hexamer and
then add another arrow from the network to the
phenotype (leave network blank for 565)
Goal Provide structural explanation for AS
patients susceptibility to aortic aneurysms!
23
Conclusions
-switch colors in order to show a6 hydroxylation
of Hyl211 -maybe add a slide about the
implications with Alports syndromeleading into
future studies

• Mass spectrometry analysis identified a1-a5 and
  a2-a6 sulfilimine-crosslinked NC1 peptides,
  providing unequivocal chemical evidence of NC1
  domain connectivities and allowing us to
  construct a homology model of the a1256 NC1
  hexamer quaternary structure
• Homology model demonstrates first heterotypic
  interaction between collagen IV protomers
• All collagen IV a-chain isoforms and all collagen
  IV networks harbor the sulfilimine bond as a
  covalent reinforcement of the hexameric NC1
  quaternary structure

24
Alports Syndrome
NEJM notes Loss of the a1256 network from the
aorta BM of Alports patients has been correlated
with an increased susceptibility to aortic
aneurysms .Thus, COL4A5 mutations underscore a
significant yet unknown structural role of the
a1256 network for vascular tissue function.
-Alports syndrome is a genetic disease which
primarily results from nonsense mutations in the
COL4A5 gene. -In AS, COL4A5 mutations lead to
loss of the a5 and a6 chains in SMC BM (aorta,
bladder tissues)led to 565 trimer, as for the
hexamer -glean useful information from
becauseanalogous situation also in AS,
immunohistochemistry studies shown that COL4A5
mutations lead to loss of the a3-a5 chains in
GBM, which is explained by failed assembly and
concomitant absence of a345 network, which leads
to nephropathy and progresses to ESRD. -So, in
an analogous mechanism, loss of the 5 chain leads
to loss of the a6 chain due to incorporation into
same collagen IV network presumed a1256
network! -Borza provided explanation, where AS
leads to concomitant absence of the a1256
networks.
Nephropathy
COL4A5 mutations
Linked to aortic abnormalities
Notes correlated with an increased
susceptibility to aortic aneurysmsthis pathology
underscores a significant yet unknown structural
role of the a1256 network.
AS underscores a distinct yet unknown structural
role for the a1256 network
25
Alports Syndrome
NEJM notes Loss of the a1256 network from the
aorta BM of Alports patients has been correlated
with an increased susceptibility to aortic
aneurysms .Thus, COL4A5 mutations underscore a
significant yet unknown structural role of the
a1256 network for vascular tissue function.
-Alports syndrome is a genetic disease which
primarily results from nonsense mutations in the
COL4A5 gene. -In AS, COL4A5 mutations lead to
loss of the a5 and a6 chains in SMC BM (aorta,
bladder tissues)led to 565 trimer, as for the
hexamer -glean useful information from
becauseanalogous situation also in AS,
immunohistochemistry studies shown that COL4A5
mutations lead to loss of the a3-a5 chains in
GBM, which is explained by failed assembly and
concomitant absence of a345 network, which leads
to nephropathy and progresses to ESRD. -So, in
an analogous mechanism, loss of the 5 chain leads
to loss of the a6 chain due to incorporation into
same collagen IV network presumed a1256
network! -Borza provided explanation, where AS
leads to concomitant absence of the a1256
networks.
COL4A5 mutations
Notes correlated with an increased
susceptibility to aortic aneurysmsthis pathology
underscores a significant yet unknown structural
role of the a1256 network.
AS underscores a distinct yet unknown structural
role for the a1256 network