MHC
STRUCTURE ANALYSIS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX
SUMMARY
1| Introduction
2| MHC class II structure
3| Labile regions
4| Non-Classical HLA
5| The peptide binding site
6| CD4 – HLA interaction
7| Conclusions
INTRODUCTION
MHC-I & MHC-II
MHC-I
MHC-II
• Expressed in all nucleated cells
• Expressed only in APCs
• Interacts with CD8 T cells and NK
• Interacts with CD4 T cells
• Presents inner antigens
• Presents outern patogenic antigens
• Involved in the control of damaged cells
• Involved in activating adaptative
immune response thru T-helper cells
T-CD8 and T-CD4 cells are not able to recognize antigens without binding to MHC proteins
MHC-II TYPES
● Classical MHC-II
antigen presentation
DR
Three types
DQ
DP
● Non-classical MHC-II
indirectly involved in antigen presentation
DM: promoter for antigenic peptide load in classical MHC-II proteins
DO: interacts with DM, modulating it’s function and the set of antigens bound to the MHC-II
GENE STRUCTURE
FOLDING AND PROCESSING
1. Folding in the RER. Binding to the unvariant chain
2. Modifications at golgi’s system
3. Transportation in vesicles
4. Unvariant chain is degradated to CLIP
5. Fusion with the lysosome. Antigenic peptide load.
6. Transportion to the membrane
PROTEIN FAMILY (SCOP)
MHC CLASS II STRUCTURE
STRUCTURE
DOMAINS: PEPTIDE BINDING GROOVE
DOMAINS: IG-LIKE
COMPARISON OF MHC-I & MHC-II STRUCTURES
MHC-I
MHC-II
COMPARISON OF MHC-I & MHC-II STRUCTURES
MHC-I
MHC-II
In MHC-II we find:
● Open ends
● Longer peptides (13-18 residues)
● Anchor residues all over the cleft
● Peptides at constant elevation
MHC-II PHYLOGENY
MHC appears 460-540 million years ago in a jawed vertebrate common antecessor
Genes for MHC class I and class II appear since the common antecessor
LABILE REGIONS
HLA-II STRUCTURES COMPARISON
| Structural superimpositions
| 4 superimpositions:
Mixing all classical HLA-II
Only DR
Only DQ
Only DP
| Alignment of the obtained structures
HLA-II SUPERIMPOSITION
3 HLA-II DR
3 HLA-II DQ
2 HLA-II DP
Sc = 8.80
RMSD = 0.86
HLA-II SUPERIMPOSITION
HLA-II DR
Sc = 9.35
RMSD = 0.81
HLA-II DQ
Sc = 9.14
RMSD = 0.49
HLA-II DP
Sc = 9.70
RMSD = 0.84
VARIABLE REGIONS
● β2 Ig-like domain
● Helical kink on the β1 domain
● 310 helix on the α chain
IG-LIKE Β2 DOMAIN
10 º
IG-LIKE Β2 DOMAIN
A-B loop
IG-LIKE Β2 DOMAIN
Sequence alignment (A-B loop)
A-B
loop
Β1-HELICAL KINK
● From β62 to β70
● Involved in conformational changes
● Higher desviation at βAsp-66
Β1-HELICAL KINK
HLA-II DR proteins show a higher desviation at βAsp 66
HLA-II DR
HLA-II DQ
HLA-II DP
Β1-HELICAL KINK
The β1-helical kink changes it’s conformation in response to
interactions with other proteins
1H15: cristal contact
1ZGL: TCR
Β1-HELICAL KINK
Sequence alignment
310 HELIX
● 3 residues/turn
● H bonds every i and i + 3 residues
● Unestable structure: RARE
310 HELIX
HLA-II DQ proteins show a higher desviation at the 310 helix
HLA-II DR
HLA-II DQ
HLA-II DP
310 HELIX
Glycines on positions α52 and α53 increase structural lability of the 310 helix
Increased affinity for HLA-DM
310 helix unwinds and rotates 20 º for
allowing interaction with MHC-DM
Glicines
3PL6
1UVQ
310 HELIX
Sequence alignment
310 HELIX
Deletions on α52 reduce affinity for HLA-II DM
DQA*02
DQA*04
HLA-DM
deficient
interaction
Autoinmune disease asociation:
Celiac disease
Diabetis mellitus 1
Multiple sclerosis
Insertion of a Gly on position α53 restores DQA*02 – MHC-DM interactions
NON-CLASSICAL HLA-II
NON-CLASSICAL HLA-II
HLA-II DR
HLA-II DM
HLA-II DO
NON-CLASSICAL HLA-II
Superimposition with classical HLA-II
3C5J (DR)
2BC4 (DM)
4I0P (DO)
NON-CLASSICAL HLA-II
Sequence is not conserved
NON-CLASSICAL HLA-II
Non-clasical HLA-II proteins seem to be remote paralogs of classical MHC-II proteins
Different sequence
Different function
Same structure
THE PEPTIDE BINDING SITE
BINDING GROOVE
● α1 and β1 domains contributing approximately equal
halves
2 a-helices flanking an 8-stranded β-sheet floor
● MCH class II is open ended
Variable length allowed
Peptide protrusion
Non anchoring of C and N terminal
BINDING POCKETS
● Constituted by a set of residues of α1 and β1
domains
● Principal pockets bind P1, P4, P6 and P9
Additional contribution of P3, P7 and P10
HLA-DR2a binding MBP 84-104
P1: Phe92
P4: Ile95
P6: Thr97
P9: Thr100
BINDING SPECIFICITY - HLA-DR2a vs. HLA-DR2b
Polymorphic
MHC class II proteins
Different architecture, charge
and shape of binding pockets
Different
binding specificity
HLA-DR2 haplotype includes two isotypes
• co-expression of two DR β chains:
DRB1*1501 and DRB5*0101
• Same α-subunit DRA*0101
Both isotypes can present
Myelin Basic Protein (MBP)
HLA-DR2a (DRA*0101,DRB5*0101)
HLA-DR2b (DRA*0101, DRB1*1501)
P1 POCKET
Phe24α, Ile31α, Phe32α, Trp43α, Ala52α, Phe54α, Asn82β, Val85β, xxx86β and Phe89β
HLA-DR2a
Gly86β – preference for bulky aromatic
residues such as Phe92 of MBP
HLA-DR2b
Val86β - preference for smaller aliphatic residues
such as Val89 of MBP
P1 POCKET
Phe24α, Ile31α, Phe32α, Trp43α, Ala52α, Phe54α, Asn82β, Val85β, xxx86β and Phe89β
HLA-DR2a
Gly86β – preference for bulky aromatic
residues such as Phe92 of MBP
HLA-DR2b
Val86β - preference for smaller aliphatic residues
such as Val89 of MBP
P4 POCKET
Gln9α, Asn62α, Tyr13β, Tyr26β, xxx71β, Ala74β, and Tyr78β
HLA-DR2a
Arg71β – preference for relative small
aliphatic residues such as Ile95 of MBP
HLA-DR2b
Ala71β - preference for large hydrophobic residues
such as Phe92 of MBP (which is P1 anchor in HLA-DR2a)
P4 POCKET
Gln9α, Asn62α, Tyr13β, Tyr26β, xxx71β, Ala74β, and Tyr78β
HLA-DR2a
Arg71β – preference for aliphatic residues
such as Ile95 of MBP
HLA-DR2b
Ala71β - preference for large hydrophobic residues
such as Phe92 of MBP (which is P1 anchor in HLA-DR2a)
PEPTIDE SHIFT
Pocket 6
Thr97
Asn94
Pocket 1
Phe92
Val98
Pocket 4
Ile95
Phe92
Pocket 9
Thr100
Thr97
3-position shift between MBP 84-102 (green and blue) and MBP 8599 (orange and purple)
Superimposition of HLA-DR2a/MBP84-102 and
HLA-DR2b/MBP85-99
BOND NETWORK
● Hydrogen bond network highly conserved
● Stereotyped mode of binding across the spectrum of peptide-MCHII interactions
MBP residues form a total of 12
hydrogen bonds with seven
residues of the HLA-DR2
complex
BOND NETWORK
● Hydrogen bond network highly conserved
● Stereotyped mode of binding across the spectrum of peptide-MCHII interactions
Contact interactions between
MBP residues and HLA-DR2
complex
v
v
DEIFHVD
v
v
v
Β sheet
a helix
310 helix
Β sheet
a helix
HLA-DR – CD4 INTERACTION
Hypervariable chains
Coreceptor
CD45
DISSOCIATION CONSTANTS
STRUCTURE
KD
General cell surface molecules
1-100µM
CTLA-4 and CD80
0.2 µM
Human CD8 binding to mouse MHC I
10µM
Human CD8 binding to HLA I
200µM
Human CD4 binding to mouse MHC II
200µM
Human CD4 binding to HLA II
>2mM
.
T-cells as a scan with high speed and sensitivity
CD4 CORRECEPTOR
● Four Ig-like domains
● D1 binds MHC II (N-term)
● D2 is important in folding
CD4 INTERACTION SITES
● 11 interaction sites with β2 and α2 domains
● Mutagenesis test
● Most important: Threonine 45 Tryptophan
● Second mutation: Glutamine 40 Tyrosine
EXPERIMENT
CD4-DM
T45W and Q40Y
Kd 8.8 µM
T45W >> Q40Y, P48L > K35P, F43L > G47S > K46R, S60R, D63R > S42G > L44T
SUPERIMPOSITION OF CD4 WT – CD4 DM
Close similarity
Sc = 8.93
RMS = 0.95
SUPERIMPOSITION OF CD4 WT – CD4 DM
Particular residues mutated
Gln
Tyr
Thr
Trp
11 residues contact 14 HLA-DR1 residues
(hydrophobic interactions)
Region 2 – alpha helix (α2 contact)
Region 1 – beta sheet (β2 contact)
REASONS FOR THE INTERACTION
BETWEEN CD4-HLADR1
HIDROGEN BONDS
Lys46 – Ser144
CD4 close to DR1
HIDROGEN BONDS
VAN DER WAALS INTERACTIONS (REGION 1)
Pro48 – Val142
Leu44 – Ser144
VAN DER WAALS INTERACTIONS (REGION 1)
Lys35 – Glu 162
Phe43 – Thr145/Ile148/Leu158
PHE 43 HYDROPHOBIC POCKET
PHE 43 HYDROPHOBIC POCKET
VAN DER WAALS INTERACTIONS (REGION 2)
Arg59 – Glu88
Asp63 – Lys176
SUPERIMPOSITION FREE-HLA vs HLA-CD4DM
Sc = 9.39
RMS = 0.70
REASONS FOR INCREASED AFFINITY
CD4 DEEP GROOVE
In the wild-type
CD4– HLA-DR1
complex, there
exists a deep
groove on the
CD4 side
Mutation 1  Threonine 45 Triptophan
Trp 45 side chain makes hydrophobic contacts with β2 Val 143
GLN40 TYR
Second mutation: Glutamine 40 Tyrosine
GLN40 TYR
Second mutation: Glutamine 40 Tyrosine
ALPHA CHAIN
ALIGNMENTS
BETA CHAIN
ALIGNMENTS
BETA CHAIN
ALIGNMENTS
CONCLUSIONS
CONCLUSIONS
• Despite the fold is conserved sequence variability in MHC II protein is in the peptide
binding site.
• The beta 2 Ig-like domain is one of the most labile regions in MHC II. It makes sense
for it’s function.
• The first helical kink at the alpha helix from beta 1 domain is involved in
conformational changes related to antigenic peptide binding and TCR interactions.
• The 3 10 helix flexibility is necessary for correct interaction with MHC II DM molecules
• Non-classical MHC-II seem to be remote paralogs of classical ones.
• All HLA-CD4 contacting residues are conserved across all HLA types
• CD4 engages in the same way HLA-DP, DQ and DR
• Increased affinity confers no survival advantage probably to avoid autoimmunity
BIBLIOGRAPHY
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Painter CA, Stern LJ. Conformational variation in structures of classical and nonclassical MHCII proteins and
functional implications. Immunol Rev. 2012; 250(1): 144-157.
Kulski JK, Shiina T, Anzai T, Kohara S, Inoko H. Comparative genomic analysis of the MHC: the evolution of class I
duplication blocks, diversity and complexity from shark to man. Immunol Rev. 2002; 190: 95–122.
Otha Y, Okamura K, McKinney EC, Bartl S, Hashimoto K, Flajnik MF. Primitive synteny of vertebrate major
histocompatibility complex class I and class II genes. PNAS. 2000; 97(9): 4712-4717.
Owen JA, Punt J, Stanford SA. Kuby inmunología. 7ª Ed. Mexico: McGraw Hill; 2014.
Li Y, Li H, Martin R, Mariuzza R. Structural basis for the binding of an immunodominant peptide from Myelin Basic
Protein in diferent registers by two HLA-DR2 proteins. JMB. 2000; 304: 177-188
Smith K, Pyrdol J, Gauthier L. Crystal structure of HLA-DR1 (DRA*0101, DRB1*1501) complexed with a peptide
from human Myelin Basic Protein. J. Exp. Med. 1998; 188: 1511-1520
Davis JS, Ikemizu S, Evans JE, Fugger L, Bakker RT, Van der Merwe PA. The nature of molecular recognition by T
cells. Nat immunol. 2003; 4: 217-24
Cole DK, Pumphrey NJ, Boulter JM, Sami M, Bell JI, Gostick E, Price DA, Gao GF, Sewell AK, Jakobsen BK. Human
TCR-binding affinity is governed by MHC class restriction. J Immunol. 2007; 178(9):5727-34
Janeway CA Jr. The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45
in T cell activation. Annu Rev Immunol. 1992;10:645-74
Wang JH, Meijers R, Xiong Y, Liu JH, Sakihama T, Zhang R, Joachimiak A, Reinherz EL. Crystal structure of the
human CD4 N-terminal two-domain fragmentcomplexed to a class II MHC molecule. Proc Natl Acad Sci U S A.
2001;98(19):10799-804
Wang XX, Li Y, Yin Y, Mo M, Wang Q, Gao W, Wang L, Mariuzza RA. Affinity maturation of human CD4 by yeast
surface display and crystal structure of a CD4-HLA-DR1 complex. Proc Natl Acad Sci U S A. 2011;108(38):15960-5
PEM QUESTIONS
The MHC:
a) Has four extracellular domains
b) Is called H-2 in human
c) The stability doesn’t depends on the structure bounded to it
d) Type I has only one alpha domain
e) Class I is very different from MHC class II
About the MHC proteins is true that:
a) MHC molecules are only present in mammals
b) Polimorphic variation tends to accumulate on the Ig-like domain.
c) MHC class II non classical molecules show low sequence identity with classical MHC molecules.
d) Humans have an enormous set of MHC class II proteins
e) Most of the MHC cristalized structures contain the transmembrane domain
Which is the correct answer about the β2 domain on the MHC-II molecules?
a) Some MHC structures miss this domain
b) This domain shows an angle of 50º of rotation
c) It’s involved in antigen presentation
d) It’s a beta + alpha fold
e) The most variable structure in the region is the A-B loop.
Which of the next proteins is known to interact with the helical kink from the β1 domain of the MHC-II?
a)CD4
b)CD8
c)TCR
d)KIR
e)TLR-4
The non-classical types of MHC class II have:
a) Different structure, different sequence and different function than the classical MHC-II.
b) Different structure, same sequence and same function than the classical MHC-II.
c) Different structure, different sequence and same function than the classical MHC-II.
d) Same structure, same sequence and same function than the classical MHC-II.
e) Same structure, different sequence and different function than the classical MHC-II.
The peptide binding groove of the MHC class II molecules:
a) Not allow peptides with variable length.
b) Binds the presented peptide with quite low specificity.
c) Have a high conserved sequence within all the types of MHC-II classical and non-classical.
d) Is composed by only residues of the alpha domain.
e) Binds the presented peptide with covalent bonds.
The TCR molecules:
a) Have three main components: the hypervariable chains, the coreceptor and an tyrosine phosphatase
b) The TCR doesn’t needs any help in order to maximize the immune response
c) The TCR uses ten molecules each time to do the signal transduction
d) Are not from immune response
e) Always needs a co-receptor in order to make the signal transduction
Which is the animal, that lives nowadays, with the simplest MHC gene structure?
a)Sharks
b) Jelly-fish
c) Dogs
d) Bony fishes
e) Snails
The coreceptor CD4:
a) Binds only MHC I molecules
b) Binds only MHC II molecules
c) Doesn’t participate in immune response
d) Has the same dissociation constant than CD8
e) Binds with a really high affinity with the MHC
In order to interact CD4 and HLA:
a) There are only hydrogen bonds making the interaction
b) Only two residues of each structure are the contacting residues
c) The interactions are in the alpha 1 and beta 1 domains of HLA
d) Both structures make disulphide bonds between them
e) There are two regions of CD4 in contact with HLA
THANK YOU FOR
YOUR ATTENTION!
PROTEIN FAMILY (CATH)
DISULPHIDE BONDS
POLIMORFIC VARIATION
Polimorphic changes tend to appear on the peptide binding site
Chain α
Chain β
Conservation
Variation