The document discusses the structure and function of the human small heat shock protein HSPB5. It summarizes that HSPB5 forms a polydisperse oligomer ranging from 10-32 subunits. The conserved alpha-crystallin domain (ACD) of each subunit was studied using NMR. The NMR data showed that at physiological pH, the ACD exists in a dimer-monomer equilibrium. Lowering the pH shifts this equilibrium toward the monomer. A single histidine residue was found to control the dimer-monomer transition. Mutation of this residue yielded a monomeric form of ACD that was shown to be a more effective chaperone than the wild type, potentially by exposing more hydrophobic

1. University of Delaware
Department of Chemistry
12/7/2015
Excited States and Conformational
Rearrangement: What do they tell us about
Chaperone Action?

2. Stress Chaperones

3. Human HSPB5
 sHSPs are found in all kingdoms and serve as ATP-
Independent chaperones.
 Human HSPB5 originally found in the
eye-lens is associated with cataracts
and myopathies.
 Also overexpressed in breast cancers; implicated in
the development of chemo-resistance
 HSPB5 overexpressed in neuro-degenerative
diseases such as Alzheimer’s

4. HSPB5: A Challenge for Structure Determination
Aquilina et al., (2003). PNAS
 Methods of Structure Determination: Crystallography and
NMR
 HSPB5: Polydisperse oligomer
 Tandem Mass Spectrometry : Oligomers ranging in size from
24-33 subunits coexist in equilibrium.
 ~ 580 kDa; Each subunit = 20 kDa
 Lower levels of
oligomers ranging in
size from 10-mers to
40-mers are also
found.

5. Method of Structure Determination
 MAS solid-state NMR
 Solution-state NMR
 Electron Microscopy (EM)
 Small Angle X-ray Scattering (SAXS)
 Rigid body Energy minimization

6.  Correlations between NMR active nuclei , 1H, 13C, and 15N:
distance restraints between residues in a protein
 Solid-state NMR: 13C or 15N detected.
 Solution-state NMR : 1H detected.
 Multimers: Distinguish between intra- and inter subunits
restraints.
15N 13C+ NHHCINTER
INTRA 15N 13COR
Mix 20% labeled with
80% unlabeled

7. Jehle and Oschkinat

8. Regions of NHHC Spectra : Inter-Subunit Correlations.

9. PSEUDO-ATOMIC MODEL OF HSPB5
Jehle et al. (2011). PNAS
Peschek et al. (2009). PNAS
24 subunits were
placed in EM-density
map and energy
minimized.

10. Hierarchical Assembly of the Oligomer
N
C

11. Substrate Binding Regions are Buried.
Misfolded
Protein
A peptide
(Alzheimer’s)
• N-terminal regional
binds misfolded
proteins.
• A peptide binds a
groove in ACD.
Mainz et al.,
(2015). NSMB

12. Is a conformational rearrangement
of HSPB5 oligomer necessary to
expose substrate-binding regions to
substrates?

13. Cellular stress and pH
 Acute cellular stresses associated with acidosis.
 pH in mouse brain following ischemic stroke drops to
pH 6.5 from pH 7.0. Shown by CEST NMR (McVicar et
al., 2014)
 Decrease in cellular pH can lead to
destabilization/decrease in solubility of some
proteins.
 Investigate HSPB5 at pH 6.5

14. 13C-13C PDSD spectra.
I133 & T134 in ACD
Groove
ssNMR Spectra Show Evidence of a Different
Conformation at pH 6.5.

15. In lieu of spending another 5
years, using vast amounts of
NMR time and saving money
for us and NIH….
Structural Biologists’ favorite
tool:
DIVIDE and CONQUER!

16. A Conserved Domain Suitable for NMR Studies
ACD
 20 kDa homodimer in solution
 ACD is conserved in all sHSPs

17.  NOEs & RDCs & chemical shifts: RosettaOligomer
 Homodimer: 6-stranded -sheet in each protomer
 Dimer interface: 10-residue -strand
SOLUTION STRUCTURE OF HSPB5-ACD
Solution structure of the ACD is similar to the structure of the ACD
dimer in the oligomer.
pH 7.5, 22 °C

18. Protein Dynamics from Solution State NMR.

19. Lineshapes in NMR Spectra
CPMG Experiments

21. CPMG Experiments Show that ACD has a minor conformation at
pH 7.5.
Map of residues showing
exchange at pH 7.5, 22 °C
• A contiguous region at the dimer
interface shows exchange implying a
concerted motion in that region.
• Dimer-Monomer equilibrium?

22. Concentration Dependence of Exchange Rate
(ACD)2 2(ACD)
kex = kdm + kmd[ACD]
kdm
kmd
• kex varies with concentration: Exchange is due to dimer-monomer
equilibrium.
• Analysis of CPMG data: pB ~5%
• KD from ITC shows that [monomer] is ~4%
Residue [ACD] (mM) kex (s-1)
119 0.7 ~1119
119 0.2 ~612
122 0.7 ~1163
122 0.2 ~100
The minor conformation at pH 7.5 and 22 °C is a monomer.

23. ACD and pH
1H-15N TROSY of ACD at pH 7.5 and pH 6.5
Peak doubling observed at pH 6.5 indicates the presence of two
conformations.

24. The Second Conformation at pH 6.5 is the Monomer
KD from ITC
pH KD (mM)
7.5 ~0.002
6.5 ~0.030
Surface Mapping of Residues (Red) Showing Peak Doubling
Dimer interface
• Increase in KD with drop in pH:
Increase in monomer population.
• AUC (Analytical
UltraCentrifugation)and SEC (Size
Exclusion Chromatography) further
confirm presence of both dimer and
monomer populations at pH 6.5

25. The Invisible, Excited State at pH 7.5 Becomes
More Populated at pH 6.5
Map of residues showing
exchange at pH 7.5
Residues showing peak
doubling at pH 6.5
B calc  B exptl
B(ppm)

26. • Dimer-monomer transition occurs in the pH range 7.5-6.5
• Imidazole rings of Histidines titrate in this range.
Residues Responsible for Dimer-Monomer Transition.
• Positively charged histidine can be mimicked by
positively charged lysine.

27. Histidines in ACD
180°
 A total of 10 histidines in ACD; 5 per protomer
 Mutated to Lysine

28. H104K-ACD is a Monomer
Regions 1H-15N HSQC Spectra
ACD at pH 6.5
H104K-ACD
15N(ppm)
AUC confirms
H104K-ACD is a
monomer.

29. How is the activity of oligomeric HSPB5
affected by the mutation H104K?

30. H104K-HSPB5 is a Better Chaperone
Model substrate: Lactalbumin (Lac)
Lac destabilized Lac aggregates
(scatter light)
DTT
Activity of HSPB5 monitored by its ability to delay scattering
H104K delays
scattering.

31. What makes H104K-HSPB5 a better
chaperone?
Investigate size and morphology by
SEC-MALS and negative-stain EM.

32. H104K forms assemblies similar to WT at pH6.5
SEC-MALS Negative Stain EM
WT
(pH 7.5)
WT
(pH 6.5)
465 kDa 720 kDaMW
# subunits ~24 ~36
<“diameter”>
H104K
(pH 7.5)
810 kDa
~41
15 (13-17) 16 (14->18) 17 (15->19)
Average Oligomer Size
Tse

33. SEC-MALS of HSPB5 and H104K-HSPB5 + Lac
SEC-MALS
Hydrodynamic Radius Hydrodynamic Radius
Tse

34. EM
Peak 1 and Peak II from SEC-MALS
Tse
H104K-HSPB5 forms smaller, longer-lived complexes
with destabilized Lac.

35. Fluorescence of ANS + H104K-HSPB5 > HSPB5
0.00E+00
1.00E+05
2.00E+05
3.00E+05
4.00E+05
5.00E+05
6.00E+05
7.00E+05
8.00E+05
9.00E+05
1.00E+06 400
411
422
433
444
455
466
477
488
499
510
521
532
543
554
565
576
587
598
609
620
631
642
653
H104K
HSPB5
Increased fluorescence :
greater hydrophobicity

36. Conclusions
• Structure of HSPB5 solved by hybrid methodology.
• The minor conformation in ACD is stabilized by the
mutation, H104K.
• H104K-HSPB5 is a better holdase, forms smaller longer
-lived complexes with denatured model substrate.
• The sampling of active conformational states and
rearrangement under stress: hallmark of sHSPs?

37. Acknowledgements
University of WA, Seattle
Rachel E.Klevit
Andrew Borst
Joseph Stout
David Fox
Amanda Clouser
Katja Dove
Scott Delbecq
Lisa Tuttle
EM and SEC-MALS,U of
Michigan
Dan Southworth
Eric Tse
Solid State NMR, FMP Berlin
Harmtut Oschkinat
Stefan Jehle
Benjamin Bardiaux
RosettaOligomer
David Baker
Nik Sgourakis
Lei Shi
NIH/NEI

38. • Dimer-monomer transition occurs in the pH range 7.5-6.5
• Imidazole rings of Histidines titrate in this range.
• Monitor the chemical shift changes of ring carbon-bound
protons and nitrogens as a function of pH. Determine pKr
values.
Residues Responsible for Dimer-Monomer Transition?

39. Protonation of a Single Histidine Favors the
Monomeric Form of ACD.
180°
6.6
?
<6<5
7.7
• pKr values shown in red
• His 83Gln: No effect
• His104Gln :Both dimer and monomer forms are observed at
pH 7.5 and 6.5
• His104Lys: Monomer at pH 7.5 and 6.5

40. Tautomeric States of Histidines in WT-ACD15Nppm
H104
H119
H111
H ppm

41. At Physiological Temperatures….
Solution State NMR performed at 22 °C to observe maximum
number of resonances.
Temperature (°C) [ACD](mM) kex (s-1)
37 0.7 1512 ±225
37 0.2 460±100
22 0.7 926±269
(ACD)2 2(ACD)
kex = kdm + kmd[ACD]
kdm
kmd

42. G = -RTlnKeq
pA = 0.096, pB = 0.04
G = 8.2 kJ mol-1
Conformational change is not
spontaneous. Stresses such as
acidosis or elevation in temperature
drive conformational change.

43.  HSPB5: ~580 kDa oligomer, 10-32 subunits.
 Each subunit: Conserved -crystallin domain
(ACD) + variable N- and C-termini.
 There are 10 human sHSPs.
 Polydisperse oligomers.
 B-Crystallin (HSPB5) seems to have evolved to
resist crystallization!
INTRODUCTION and OUTLINE
 MAS solid-state NMR and EM: model of HSPB5
oligomer
 Solution state studies on the ACD domain:
functional questions