1. Elucidating the Structure of the Inner-Ear Tip Link: An Unusual Cadherin-23 Fragment
Domenic J. Termine and Marcos Sotomayor
Department of Chemistry and Biochemistry
The Ohio State University
How Hearing Works
ReferencesAcknowledgements
The Hair-Cell Tip Link
I would like to acknowledge The Sotomayor Research Group for their continued support
and assistance in the lab. Funding for this project was provided by OSU (Undergraduate
Research Scholarship) and NIH NIDCD K99/R00 DC012534-01
Purification Under Denaturing Conditions
• The tip link is formed by two non-classical
cadherins: Cadherin-23 (Cdh-23) and
Protocadherin-15 (Pcdh-15) [3]
• Cadherins (calcium-dependent adhesion
molecules) are a class of type-1 transmem-
brane proteins with multiple extracellular (EC)
repeats
• A cdh-23 parallel dimer and a pcdh-15
parallel dimer interact tip-to-tip to form the
tip link
• Cdh-23 has 27 extracellular repeats
• Pcdh-15 has 11 extracellular repeats
Cloning of Unusual Cadherin-23 Fragments
• The DNA fragments amplified by PCR (both mouse and human) and the pET-21a(+) vector
were cut with the restriction enzymes Ndel and Xhol
32-nirehdaC
EC1
51-nirehdacotorP
EC1
EC11
EC27
F
F
Membrane
Membrane
The ear – illustrated
by Max Brödel (1939)
Stereocilia
Kinocilium
Tip link
Force
Stereocilia bundles,
the mechanosensing
organelles of the hair cells
lining the Organ of Corti
within the cochlea
Tip links
Mechanical Energy Electric Signals
• The vibrations of a sound wave enter the cochlea,
a fluid-filled organelle containing thousands of hair cells
• Each hair cell contains a small patch of stereocilia
• In response to vibration, the stereocilia rock
back and forth causing the tip link to act in series
with an elastic “gating spring” to pull open a
transduction channel [2]
• The molecular architecture of this complex is unknown
Pcdh-15
Mechanosensitive
Channel?
5 nm
13nm
Future Directions
Cdh-23
Unusual Cadherin-23 Calcium-Binding Sites
Pcdh-15 EC1+2
Cdh-23 EC1+2
• Previous experiments and molecular
dynamics simulations suggest that Ca2+
and Ca2+
-binding residues are crucial
for cdh-23 elasticity and function
• There is one non-conserved calcium
binding site between EC repeats 12 and
13 (aspartate is replaced by a serine at
position 1339)
• How does the variation from aspar-
tate to serine affect the overall strength
and structure of EC12 and the tip link?
: : . . * : . : : *
EC1 QV NRL P F F T NHF F DT Y L L I S E D- T P V GS S V T QL L A - - - - QDMDNDP L - - - - - - - - - - - V F G- V S - - - GE E A S RF F A V E P DT GV V WL - - - RQP L DRE T K S E - - - - - - F T V E F S V S DH- - - - - - - QG- V I T RK V N- - - - - - - - - - - I QV GDV NDNA P T F 109
EC2 - - - - - - - - HNQP Y S - - V RI P E N- T P V GT P I F I V N- - - - A T DP DL GA - - - - - - - - - GGS V L Y S F Q- - - - - P P S QF F A I DS A RGI V T V - - - I RE L DY E T T QA - - - - - - Y QL T V NA T DQ- - - - - - - DK T RP L S T L A N- - - - - - - - L A I I I T DV QDMDP I F 104
EC3 - - - - - - - - I NL P Y S - - T NI Y E H- S P P GT T V RI I T - - - - A I DQDK GR- - - - - - - - - P RGI GY T I V S GNT NS I F A L DY I S G- V L T L NG- - - - - L L DRE NP - L Y S HG- - F I L T V K GT E L - - - - - - - NDDRT P S DA T V T - - - - - T T F NI L V I DI NDNA P E F 112
EC4 - - - - - - - - NS S E Y S - - V A I T E L - A QV GF A L P L F I Q- - - V V DK DE NL G- - - - - - - L NS MF E V Y L V GNNS HHF I I S P T S V QGK A DI RI - RV A I P L DY E T V DR- - - - - - Y DF DL F A NE S - - - - - - - V P DHV GY A K V K - - - - - - - - - - I T L I NE NDNRP I F 112
EC5 - - - - - - - - S QP L Y N- - I S L Y E N- V T V GT S V L T V L A - - T - - DNDA G- - - - - - - - - T F GE V S Y F F S D- - - - DP DR- F S L DK DT GL I ML - - - I A RL DY E L I QR- - - - - - F T L T I I A RDG- - - - - - - GGE E T T GRV R- - - - - - - - - - - I NV L DV NDNV P T F 101
EC6 - - - - - - - - QK DA Y V - - GA L RE N- E P S V T QL V RL R- - - - A T DE DS P P - - - - - - - - - NNQI T Y S I V S A S A F GS Y F DI S L Y E GY GV I S V - - - S RP L DY E QI - S NG- - - L I Y L T V MA MDA - - - - - - - G- - - NP P L NS T - - - - - - V P V T I E V F DE NDNP P T F 110
EC7 - - - - - - - - S K P A Y F - - V S V V E N- I MA GA T V L F L N- - - - A T DL DR- - - - - - - - - - - S RE Y GQE S I I Y S L - E GS T QF RI NA RS GE I T T - - - T S L L DRE T K S E - - - - - - Y I L I V RA V D- - - - - - - - - GGV - GHNQK T GI - - - - A T V NI T L L DI NDNHP T W 107
EC8 - - - - - - - - K DA P Y Y - - I NL V E M- T P P DS DV T T V V - - - - A V DP DL GE - - - - - - - - - NGT L V Y S I QP - - P NK F Y S L NS T T GK I RT T HA - - - - - ML DRE NP DP HE A E L MRK I V V S V T DC- - - - - - - GRP P L K A T S S - - - - - - - A T V F V NL L DL NDNDP T F 112
EC9 - - - - - - - - QNL P F V - - A E V L E G- I P A GV S I Y QV V - - - - A I DL DE GL - - - - - - - - - NGL V S Y RMP V - - GMP RMDF L I NS S - S GV V V T - - - T T E L DRE RI - - - - - A - E Y QL RV V A S DA - - - - - - - GT P T K S S T - - - - - - - - - S T L T I HV L DV NDE T P T F 105
EC10 - - - - - - - - F P A V Y N- - V S V S E D- V P RE F RV V WL N- - - - CT DNDV GL N- - - - - - - - - A E L S Y F I T G- - - GNV DGK F S V GY RDA V V RT - - - V V GL DRE T T A A - - - - - - Y ML I L E A I DN- - - - - - - GP V GK RHT GT A - - - - - - - T V F V T V L DV NDNRP I F 107
EC11 - - - - - - L QS S - - Y E A - S - V P E D- I P E GHS I L QL K - - - - A T DA DE GE - - - - - - - - - F GRV WY RI L H- - - GNHGNNF RI HV S NGL L MRG- - P RP L DRE RNS S - - - - - - HV L I V E A Y - N- - - - - - - HDL GP MRS S - - - - - - - - V RV I V Y V E DI NDE A P V F 106
EC12 - - - - - - - - T QQQY S R- L GL RE T - A GI GT S V I V V Q- - - - A T DRDS GD- - - - - - - - - GGL V NY RI L S G- A E GK F E I DE S - - - T GL I I T - - - V NY L DY E T K T S - - - - - - Y MMNV S A T DQ- - - - - - - A P P F NQGF CS V - - - - - - - Y I T L L - - NE L DE A V QF 105
EC13 - - - - - - - - S NA S Y E - - A A I L E N- L A L GT E I V RV Q- - - - A Y S I DN- - - - - - - - - - - L NQI T Y RF NA Y T S T QA K A L F K I DA I T GV I T V - - - QGL V DRE K GDF - - - - - - Y T L T V V A DD- - - - - - - - GGP K V DS T - - - - - - - - - V K V Y I T V L DE NDNS P RF 105
EC14 - - - - - - - DF T S DS A - - V S I P E D- CP V GQRV A T V K - - - - A WDP DA GS - - - - - - - - - NGQV V F S L A S - - GNI A GA F E I V T T NDS I GE V - F V A RP L DRE E L - - - - - D- HY I L QV V A S DR- - - - - - - GT P P RK K D- - - - - - - - - HI L QV T I L DI NDNP P V I 109
EC15 - - - - - - - E S P F GY N- - V S V NE N- V GGGT A V V QV R- - - - A T DRDI GI - - - - - - - - - NS V L S Y Y I T E GNK DMA F RMDRI S - - - GE I A T R- - P A P P DRE RQS F - - - - - - Y HL V A T V E DE - - - - - - - G- - - T P T L S A T - - - - - - T HV Y V T I V DE NDNA P MF 107
EC16 - - - - - - - - QQP HY E - - V L L DE GP DT L NT S L I T I Q- - - - A L DL DE GP - - - - - - - - - NGT V T Y A I V A GNI V NT F RI DRH- - - MGV I T A - - - A K E L DY E I S HGR- - - - - Y T L I V T A T DQ- - - - - - - CP I L S HRL T S T - - - - - - T T V L V NV NDI NDNV P T F 110
EC17 - - - - - - - - - P RDY E GP F E V T E G- QP - GP RV WT F L - - - - A HDRDS GP - - - - - - - - - NGQV E Y S I MD- - GDP L GE F V I S P V - E GV L RV - RK DV E L DRE T I - - - A - - - F Y NL T I CA RDR- - - - - - - GMP P L S S T - - - - - - - - - ML V GI RV L DI NDNDP V - 106
EC18 - - - - - - - L L NL P MN- - I T I S E N- S P V S S F V A HV L - - - - A S DA DS - - - - - - - - - GCNA RL T F NI T A G- - - NRE RA F F I NA T T GI V T V - - - NRP L DRE RI P E - - - - - - Y K L T I S V K DN- - - - - P E NP - - RI A RRDY - - - - - - DL L L I F L S DE NDNHP L F 109
EC19 - - - - - - - - T K S T Y Q- - A E V ME N- S P A GT P L T V L NGP I L A L DA DQDI Y - - - - - - - - - A V V T Y QL L G- - - - A QS GL F DI NS S T GV V T V - RS GV I I DRE A F S P - - - P - I L E L L L L A E DI - - - - - - - GL L NS T A H- - - - - - - - - - - L L I T I L DDNDNRP T F 110
EC20 - - - - - - - - S P A T L T - - V HL L E N- CP P GF S V L QV T - - - - A T DE DS GL - - - - - - - - - NGE L V Y RI E A GA QDR- F L I HL V T - - - GV I RV G- - NA T I DRE E QE S - - - - - - Y RL T V V A T DR- - - - - - - G- - - T V P L S GT - - - - - - A I V T I L I DDI NDS RP E F 105
EC21 - - - - - - - - L NP I QT - - V S V L E S - A E P GT V I A NI T - - - - A I DHDL NP K L - - - E Y HI V GI V A K DDT DRL V P NQE DA F A V NI NT GS V MV - - - K S P MNRE L V A T - - - - - - Y E V T L S V I DN- - - - - - - A S DL P E RS V S V P N- - - - A K L T V NV L DV NDNT P QF 119
EC22 - - - - - - K P F GI T Y Y M- E RI L E G- A T P GT T L I A V A - - - - A V DP DK GL - - - - - - - - - NGL V T Y T L L D- - L V P P GY V QL E DS S A GK V I A - - - NRT V DY E E V HW- - - - - - L NF T V RA S D- - - - - - - NGS P P RA A E I P - - - - - - - - - V Y L E I V DI NDNNP I F 109
EC23 - - - - - - - - DQP S Y Q- - E A V F E D- V P V GT I I L T V T - - - - A T DA D- - - - - - - - - - - - S GNF A L - - I E Y S L GDGE S K F A I NP T T GDI Y V - - - L S S L DRE K K DH- - - - - - Y I L T A L A K DN- - - - - - P GDV - A S NRRE NS - - - - - V QV V I QV L DV NDCRP QF 107
EC24 - - - - - - - - S K P QF S - - T S V Y E N- E P A GT S V I T MM- - - - A T DQDE GP - - - - - - - - - NGE L T Y S L E GP GV E A F HV DMDS - - - - GL V T T - - - QRP L QS Y E K F S - - - - - - - - L T V V A T DG- - - - - - - G- - - E P P L WGT - - - - - - T ML L V E V I DV NDNRP V F 102
EC25 - - - - - - - V RP P NGT I - L HI RE E - I P L RS NV Y E V Y - - - - A T DK DE GL - - - - - - - - - NGA V RY S F L K T A GNRDWE F F I I DP I S GL I QT - - - A QRL DRE S QA V - - - - - - Y S L I L V A S DL - - - - - - - GQP V P Y E T MQP - - - - - - - - L QV A L E DI DDNE P L F 111
EC26 - - - V RP P K GS P QY QL - L T V P E H- S P RGT L V GNV T G- - - A V DA DE GP - - - - - - - - - - NA I V Y Y F I A A - - GNE E K NF HL QP DGCL L V L - - - - RDL DRE RE A I - - - - - - F S F I V K A S S NRS WT P P RGP S P T L DL V A DL T - - - L QE V RV V L E DI NDQP P RF 124
EC27 - - - - - - - - T K A E Y T - - A GV A T D- A K V GS E L I QV L - - - - A L DA DI GNN- S L V F Y S I L A I HY F RA L A NDS E DV GQV F T MGS MDGI - - - - - - L RT F DL F MA Y S P G- - - Y F V V DI V A RDL - - - - - - - A GHNDT A I I GI Y I L RDDQRV K I V I NE I P DRV RGF 125
1 . . . . . . . 1 0 . . . . . . . . 2 0 . . . . . . . . 3 0 . . . . . . . . 4 0 . . . . . . . . 5 0 . . . . . . . . 6 0 . . . . . . . . 7 0 . . . . . . . . 8 0 . . . . . . . . 9 0 . . . . . . . 1 0 0 . . . . . . . 1 1 0 . . . . . . . 1 2 0 . . . . . . . 1 3 0 . . . . . . . 1 4 0 . . . . . . . 1 5 0 . . . . . . .
DXNDNXEXBASE
DREDXD XDXTOP
N-TERMINUS
Ca2+
0 Ca2+
0,3Ca2+
1,2 Ca2+
1,2 Ca2+
0,3 Ca2+
1,2,3
EC1
EC2
EC3
EC4
EC5
EC6
EC7
EC8
EC9
EC10
EC11
EC12
EC13
EC14
EC15
EC16
EC17
EC18
EC19
EC20
EC21
EC22
EC23
EC24
EC25
EC26
EC27
. *
QDMDN
T DP DL
DQDK
V DK DE
DNDA
T DE DS
T DL DR
V DP DL
DL DE
T DNDV
T DA DE
T DRDS
Y S I DN
WDP DA
T DRDI
DL DE
HDRDS
S DA DS
DA DQ
T DE DS
DHDL
V DP DK
T DA D-
T DQDE
T DK DE
V DA DE
DA DI
DXD
Ca2+
0,3
EC12
EC13
EC14
T DRDS
Y S I DN
WDP DA
EC11
EC12
EC13
EC12
1
2
3
Human Mouse
EC13
EC14
Cdh-23 EC1+2
EC1
EC2
Binding (B) Buffer
20 mM Tris-HCl pH 7.5
10 mM CaCl2
20 mM Imidazole
6 M Gu-HCl
Elution (E) Buffer
20 mM Tris-HCl pH 7.5
10 mM CaCl2
500 mM Imidazole
6 M Gu-HCl
Protein Pellet
(large culture)
B Buffer Sonication
Supernatant with
desired protein and
other proteins
Ni Sepharose Beads
Protein His•Tag
Bound to Ni
Eluted Protein
W1 W2
E Buffer
Gu-HCl
Dialysis
Gu-HCl
• Determine whether the diffraction pattern (shown above) is that of mm cdh23 EC12-13 or
salt. If indeed protein, refine around the buffer condition to grow crystals more conducive to
generating a high-quality diffraction data set
• Continue to create and explore constructs centered around the non-conserved
Ca2+
-binding site between EC 12+13 to ultimately generate crystals and solve a structure
that will determine whether the alteration from aspartate to serine has some physiological
purpose
• Complete the trypsin digestion assay to analyze the aspartate-serine variation as an alter-
native and complimentary approach to structural determination
• Utilize mutagenesis and the biochemical techniques acquired thus far to analyze the
mutation D1341N, known to cause nonsyndromic deafness
Cochlear hair cells - R. Harrison & R. Mount
Bechara Kachar 2000
Adapted from [1]
Adapted from [1]
Adapted from [4]
Made with VMD [5]
[1] Sotomayor, M., Weihofen, W. A., Gaudet, R., Corey, D. P. Nature, 492 (7427): 128-132, 2012
[2] Gillespie, PG., Müller U. Cell, 139:33-44, 2009
[3] Kazmierczak, et al. Nature, 449:87-91, 2007
[4] Sotomayor, M.*, Weihofen, W.A.*, Gaudet, R., Corey, D. P. Neuron, 66:85-100, 2010
[5] Humphrey, W., Dalke, A., Schulten, K.. J. Molec. Graphics, 14.1:33-38, 1996
EC11
EC12
EC13
EC14
EC15
pET-21a(+)
Ligation Transformation
40 60 80 100mL
0
50
100
150
200
250
300
mAU
40 60 80 100mL
0
50
100
150
200
250
300
mAU
• The DNA sequence for the mouse and human constructs were sequenced and determined
to have appropriate restriction sites, histidine tag, and nucleotides
Large Scale
•LB (Lysogeny Broth)
•Ampicillin
•Chloramphenicol
•IPTG
•hsCdh23 or mmCdh23 DNA
Size Exclusion Chromatography
Dialysis Buffer
150 mM NaCl
2 mM CaCl2
20 mM Tris-HCl pH 8.0
400 mM Arginine
+
2 mM DTT (in bag)
40 60 80 100mL
0
50
100
150
200
250
300
mAU
11 1312 12 1413 1312
L D5D12C6 D2E5 D3L D1 L L D1 E3E1E2 E4D5C5 E7E6E5 E11
40kDa
30kDa 30kDa
40kDa
C6 D5D12 D2
C5
D5 E3
Mutagenesis and Trypsin Digestion Assay
hsCdh-23 EC11-13 mmCdh-23 EC12-14 mmCdh-23 EC12-13
mm cdh23 EC 12-13
L E2 E10E9E8E7E6 E11 E12
E11 E2 E8
X-Ray Crystallography
0.1 M Sodium Acetate pH 4.6
1.25 M Lithium Acetate
Wild-type (serine at position 1339)
Mutant (aspartate at position 1339)
Buffer
Buffer + protein
12
14
13
*
• The relevance of the aspartate-to-serine variation
will be tested by measuring the Ca2+
-binding affinity
of two constructs (shown to the left)
• Trypsin cleaves cadherins to a varying degree,
correlated with Ca2+
-binding affinity. The construct
with a higher affinity for Ca2+
will require a lower
concentration of additional Ca2+
to protect it from
trypsin digestion
• Expected Result 1 - both constructs produce
similar binding affinities indicating a less significant
role for the non-conserved serine at position 1339
• Expected Result 2 - the mutant exhibits a higher
affinity for Ca2+
compared to the wild-type construct
indicating a physiological purpose for the non-
conserved serine at position 1339
• The Corning® CrystalEX 96 well crystallization plate is designed for high-throughput protein
crystallization through sitting drop vapor diffusion
Example of protein diffraction mm cdh23 EC 12-13 or salt diffraction? Example of salt diffraction
(A. Jaiganesh)
• The sodium acetate/lithium acetate crystals will be sent to the Argonne National
Laboratory Synchrotron where they will be tested with an extremely powerful
source of X-rays to determine their identity
Competent Cells
20kDa
B Buffer
SEC Buffer
150 mM NaCl
2 mM CaCl2
20 mM Tris-HCl pH 8.0