OGlcNAcylation and phosphorylation have opposing structural effects in tau: Phosphothreonine induces particular conformational order

Article abstract of DOI:10.1021/ja407156m

Phosphorylation and OGlcNAcylation are dynamic intracellular protein post-translational modifications that frequently are alternatively observed on the same serine and threonine residues. Phosphorylation and OGlcNAcylation commonly occur in natively disordered regions of proteins, and often have opposing functional effects. In the microtubule-associated protein tau, hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of Alzheimer's disease, whereas OGlcNAcylation stabilizes the soluble form of tau. A series of peptides derived from the proline-rich domain (residues 174-251) of tau was synthesized, with free Ser/Thr hydroxyls, phosphorylated Ser/Thr (pSer/pThr), OGlcNAcylated Ser/Thr, and diethylphosphorylated Ser/Thr. Phosphorylation and OGlcNAcylation were found by CD and NMR to have opposing structural effects on polyproline helix (PPII) formation, with phosphorylation favoring PPII, OGlcNAcylation opposing PPII, and the free hydroxyls intermediate in structure, and with phosphorylation structural effects greater than OGlcNAcylation. For tau_196-209, phosphorylation and OGlcNAcylation had similar structural effects, opposing a nascent α-helix. Phosphomimic Glu exhibited PPII-favoring structural effects. Structural changes due to Thr phosphorylation were greater than those of Ser phosphorylation or Glu, with particular conformational restriction as the dianion, with mean ³J_αN = 3.5 Hz (pThr) versus 5.4 Hz (pSer), compared to 7.2, 6.8, and 6.2 Hz for Thr, Ser, and Glu, respectively, values that correlate with the backbone torsion angle. Dianionic phosphothreonine induced strong phosphothreonine amide protection and downfield amide chemical shifts (δ_mean = 9.63 ppm), consistent with formation of a stable phosphate-amide hydrogen bond. These data suggest potentially greater structural importance of threonine phosphorylation than serine phosphorylation due to larger induced structural effects.

Full text of DOI:10.1021/ja407156m

Article
pubs.acs.org/JACS
Open Access on 02/21/2015
OGlcNAcylation and Phosphorylation Have Opposing Structural
Effects in tau: Phosphothreonine Induces Particular Conformational
Order
Michael A. Brister,† Anil K. Pandey,† Agata A. Bielska, and Neal J. Zondlo*
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States
S
* Supporting Information
ABSTRACT: Phosphorylation and OGlcNAcylation are
dynamic intracellular protein post-translational modifications
that frequently are alternatively observed on the same serine
and threonine residues. Phosphorylation and OGlcNAcylation
commonly occur in natively disordered regions of proteins,
and often have opposing functional effects. In the microtubule-
associated protein tau, hyperphosphorylation is associated with
protein misfolding and aggregation as the neurofibrillary tangles of Alzheimer’s disease, whereas OGlcNAcylation stabilizes the
soluble form of tau. A series of peptides derived from the proline-rich domain (residues 174−251) of tau was synthesized, with
free Ser/Thr hydroxyls, phosphorylated Ser/Thr (pSer/pThr), OGlcNAcylated Ser/Thr, and diethylphosphorylated Ser/Thr.
Phosphorylation and OGlcNAcylation were found by CD and NMR to have opposing structural effects on polyproline helix
(PPII) formation, with phosphorylation favoring PPII, OGlcNAcylation opposing PPII, and the free hydroxyls intermediate in
structure, and with phosphorylation structural effects greater than OGlcNAcylation. For tau196−209, phosphorylation and
OGlcNAcylation had similar structural effects, opposing a nascent α-helix. Phosphomimic Glu exhibited PPII-favoring structural
effects. Structural changes due to Thr phosphorylation were greater than those of Ser phosphorylation or Glu, with particular
conformational restriction as the dianion, with mean 3JαN = 3.5 Hz (pThr) versus 5.4 Hz (pSer), compared to 7.2, 6.8, and 6.2 Hz
for Thr, Ser, and Glu, respectively, values that correlate with the backbone torsion angle ϕ. Dianionic phosphothreonine induced
strong phosphothreonine amide protection and downfield amide chemical shifts (δmean = 9.63 ppm), consistent with formation of
a stable phosphate-amide hydrogen bond. These data suggest potentially greater structural importance of threonine
phosphorylation than serine phosphorylation due to larger induced structural effects.
INTRODUCTION
■
Genome sequencing has revealed that most higher eukaryotes
have a relatively limited set of genes, whose numbers do not
correlate with organismal complexity.1 The ability of a limited
number of genes to achieve diverse protein functions depends
on a series of post-translational modifications (PTMs),
including phosphorylation, glycosylation, acylation, methyla-
tion, lipidation, protein ligation, sulfation, and myriad
oxidations, that result in controllable and conditional functions
of proteins. Intracellularly, serine and threonine residues are
modified by phosphorylation, regulated by protein kinases and
phosphatases that collectively account for 2.5% of human
Figure 1. Intracellular post-translational modifications of Ser and Thr
genes, and by OGlcNAcylation, controlled by OGlcNAc
transferases and OGlcNAcases (Figure 1).2 Frequently, the
by phosphorylation and OGlcNAcylation. Glu is a common pSer/
pThr mimic. The diethylphosphate triester of Ser/Thr is neutral and
same residues that are phosphorylated are also observed under
sterically similar to OGlcNAc.
different conditions to be OGlcNAcylated. Interestingly, in
many cases, phosphorylation and OGlcNAcylation are observed
fibrils and precipitate as the major protein components of the
to have opposing functional effects. Thus, improved under-
neurofibrillary tangles (NFTs) observed in Alzheimer’s disease
standing of the differential structural effects of phosphorylation
and other neurodegenerative disorders, including frontotem-
and OGlcNAcylation is of broad potential application in
poral dementia, Pick’s disease, and chronic traumatic
cellular biology.
The protein tau is a 441 amino acid (largest isoform) natively
disordered microtubule-binding protein that is most prominent
in neurons. Hyperphosphorylated forms of tau aggregate as
Received: July 12, 2013
Published: February 21, 2014
© 2014 American Chemical Society
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encephalopathy (CTE) (collectively termed tauopathies).3 The
protein tau consists of a number of functional domains (Figure
2), including 4 hydrophobic tubulin-binding domains (TBDs)
including tau, indicate that conformations observed in peptides
are similar to those observed in the larger protein contexts,
because of the absence of stable tertiary and quaternary
structures.5 Because of the challenges of both structure
determination in natively disordered proteins and of reliable
preparation of homogeneous samples of expressed proteins
with defined patterns of multiple protein post-translational
modifications, particularly for OGlcNAcylation, we have used
peptide models to understand the local structural effects of
natively disordered regions of proteins.6
We previously investigated the structural effects of
phosphorylation on peptides derived from the proline-rich
domain of tau.7 In that work, we found that phosphorylation of
tau peptides induced a structural change promoting polyproline
helix (PPII). OGlcNAcylation of tau, which has been identified
on a series of sites in the proline-rich domain that are also sites
of phosphorylation, has been found to be protective against
hyperphosphorylation and neurofibrillary tangle formation.8
Indeed, inhibitors of OGlcNAcase, the enzyme that removes
the OGlcNAc group and thus can functionally inhibit
phosphorylation by preventing access of the kinase substrate,
are under investigation as potential therapeutics in Alzheimer’s
disease.9 The mechanism of OGlcNAcylation-mediated pro-
tection against NFT formation could be via prevention of
phosphorylation, and additionally or alternatively OGlcNAcy-
lation could promote a structure change that is different than
that of phosphorylation.
Figure 2. Top: Schematic of the primary sequence and functional
domains of tau. Red, hydrophobic regions A and B; green, proline-rich
domain (PRD); blue, tubulin-binding domain (TBD) repeats 1−4
(R1, R2, R3, R4). The most commonly used boundaries of the TBDs
are indicated; notably, residues 242−251 of R1 have poor homology to
analogous residues in R2, R3, and R4 thus defined. The TBD
boundaries have alternatively been defined as R1 residues 252−282,
R2 283−313, R3 314−344, R4 345−376. By this definition, the
proline-rich domain extends through proline 251. Most phosphor-
ylation sites associated with tau aggregation in Alzheimer’s disease are
in the proline-rich domain and in the C-terminal domain. Additional
phosphorylation sites are in the N-terminus and in the TBDs. Bottom:
Sequences of tau-derived proline-rich peptides examined in this study.
All peptides were acetylated at the N-terminus and contained C-
terminal amides. Residues in blue indicate sites modified by
phosphorylation, diethylphosphorylation, or OGlcNAcylation. Resi-
dues in red correspond to the key residues of the hydrophobic B motif.
Residue numbers are based on the largest (441 residue) isoform of tau.
N-terminal tyrosines were added to tau211−219 and tau229−238 for
concentration determination. In contrast to these peptides, addition of
either an N-terminal or C-terminal Tyr to tau174−183 resulted in
substantial changes to its CD spectrum.
RESULTS
■
In view of the general observation of phosphorylation and
OGlcNAcylation occurring on similar sites, particularly within
natively disordered regions of proteins, we sought to examine
within a biologically relevant sequence context the relative
structural effects of phosphorylation and OGlcNAcylation
compared to the unmodified Ser/Thr residues. A series of
peptides derived from the proline-rich domain of tau was
synthesized, and their structures were analyzed as free
hydroxyls and as phosphorylated and OGlcNAcylated amino
acids, by circular dichroism and NMR (Figure 2). All residues
that contained post-translational modifications have been
previously identified as sites of these post-translational
modifications in tau.8,10
(residues 242−367) that are directly responsible for both
binding microtubules and for tau aggregation, an N-terminal
hydrophobic region which dynamically interacts with the
TBDs, and a proline-rich domain (residues 174−241), which
also contains a second hydrophobic region (residues 220−
231).4 The proline-rich domain of tau serves as a linker
between the N-terminal sequence and the tubulin-binding
domains. The majority of phosphorylation sites identified to be
important to hyperphosphorylation-mediated tau aggregation
are in the proline-rich domain, with additional phosphorylation
sites located C-terminal to the tubulin-binding domains in a
region that also interacts with the TBD to stabilize it.
Knowledge of the structural effects of post-translational
modifications of tau is important to understand the
mechanisms of pathological protein misfolding induced by
hyperphosphorylation observed in the Alzheimer’s diseased
brain, as well as to understand how to maintain tau in a soluble,
nonaggregated form. Data on natively disordered proteins,
The protected Fmoc β-OGlcNAc serine and threonine
amino acids were synthesized via a modification of the
methodology of Arsequell et al. (Scheme 1).11 The protected
OGlcNAcylated amino acids were incorporated in peptides, and
the peptides were subjected to TFA cleavage/deprotection and
purified by HPLC. The purified peptides containing protected
OGlcNAc hydroxyls were then subjected to deesterification via
NaOMe/MeOH and purified to generate peptides with defined
patterns of OGlcNAcylation on multiple residues (Scheme
2).12
Scheme 1. Synthesis of OGlcNAcylated Fmoc Amino Acids via a Modification of Ref 11
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this band can be obscured by the substantially more intense
signal from α-helices ([θ]max ∼ −33 000 deg cm2 dmol−1 for α-
helix, compared to [θ]max = +5000 deg cm2 dmol−1 for
polyproline helix); thus, even nascent α-helices and β-structure
can substantially obscure the CD signal of polyproline helix in
larger peptides. PPII is also characterized by a negative band at
∼200 nm, although other structures can contribute to the
intensity of this band, and thus analysis of this band is
nonquantitative. Polyproline helix can also be identified by
changes in the λmax in CD and in NMR via coupling
constants.16 In general, it is difficult to identify polyproline
helix in larger peptides and proteins by either CD or NMR
because of similarities to data in random coil, though
polyproline helix can be definitively identified and is observed
to thermally melt to a random coil conformation, which has a
different CD signature. Because of these complications, smaller
peptides often have substantial advantages for the definitive
identification of polyproline helix.
In polyproline helix propensity scales, serine and threonine
have low PPII propensities, due to the possibility of multiple
side chain/main chain hydrogen bonds and χ1 conformational
heterogeneity.17 The lowest PPII propensities are observed for
aromatic amino acids and for sterically hindered β-branched
amino acids (Thr, Ile, Val, and particularly the highly sterically
congested tert-leucine (Tle)), indicating that steric hindrance
near the protein backbone strongly opposes PPII.
All peptides were analyzed by circular dichroism. In tau174−183
(Figure 3), which contains the Alzheimer’s disease phosphoe-
pitope pThr175/pThr181, the phosphorylated peptide was
observed to have greater PPII (larger signal at 228 nm, a
defined maximum indicative of PPII) than the nonphosphory-
lated peptide.7,15a,18 Interestingly, the CD of the phosphory-
lated peptide was similar to the peptide with both threonines
changed to the phosphomimic (and PPII-favoring residue) Glu,
although the structural effect of replacement of Thr by Glu was
less than that of Thr phosphorylation (Figure 3b). In contrast,
the OGlcNAcylated peptide exhibited reduced PPII compared
to the nonphosphorylated peptide, indicating that OGlcNAcy-
lation disfavors PPII.19 The effect of diethylphosphorylation
was qualitatively similar to that of OGlcNAcylation, though
greater in magnitude. The structural change of OGlcNAcylation
was also observable in a red shift of the λmax of the CD
Scheme 2. Synthesis of OGlcNAcylated Peptides via Initial
Purification of the Peracetylated Peptides Followed by
Deacetylation under Basic Conditions
In contrast to the common use of Glu as a mimic of
phosphoserine/phosphothreonine, there is no readily accessible
mimic of OGlcNAcylated serine/threonine. The diethylphos-
phate modification of serine and threonine, which is readily
incorporated into peptides using phosphoramidites by standard
chemistry employed for peptide phosphorylation, generates a
derivatized side chain that is neutral and sterically similar to
OGlcNAc. However, in contrast to OGlcNAcylated Ser/Thr,
which typically require expensive amino acids (Fmoc-Ac3−
SerOGlcNAc and Fmoc-Ac3−ThrOGlcNAc cost $600/100 mg,
a quantity sufficient for the incorporation of just one
OGlcNAcylated residue in a single peptide synthesized at
small scale) and/or substantial synthetic manipulation, peptides
with the diethylphosphate modification are readily synthesized
on solid phase from inexpensive, commercially available
reagents (Figure 1). Diethylphosphate, which exhibits sub-
stantial steric effects in model peptides,13 is thus potentially a
practical mimic of the steric effects of OGlcNAcylation.
Therefore, in addition to synthesizing the OGlcNAcylated
peptides, we also synthesized peptides with the diethylphos-
phates at sites of OGlcNAcylation to investigate them as
potential readily accessible mimics of OGlcNAcylation.
Polyproline helix (PPII) is a predicted major conformation of
protein proline-rich domains.14 The PPII content in peptides
can be quantified using circular dichroism (CD), via the weak
positive band at ∼228 nm.15 In larger peptides, the intensity of
Figure 3. CD spectra of tau174−183 peptides (Ac−KTPPAPKTPP-NH2) in water with 5 mM phosphate buffer and 25 mM KF at pH 7.5. (a) Thr,
green squares; phosphothreonine, red circles; ThrOGlcNAc, blue diamonds; ThrOPO3Et2, black triangles. (b) Peptides with Thr (green squares) or
with both Thr residues replaced by either Glu (Ac−KEPPAPKEPP-NH2) (magenta open circles) or tert-leucine (Tle) (Ac−KTlePPAPKTlePP-
NH2) (purple open diamonds).
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Figure 4. CD spectra of (a) tau211−219, (b) tau229−238, and (c) tau196−209 peptides in water with 5 mM phosphate buffer and 25 mM KF at pH 7.5.
Unmodified Ser/Thr, green squares; phosphoserine/phosphothreonine, red circles; Ser/Thr OGlcNAc, blue diamonds; Ser/Thr OPO3Et2, black
triangles.
minimum, from 201 nm in the phosphorylated peptide to 203
nm in the OGlcNAcylated peptide and to 204 nm in the
diethylphosphorylated peptide, as well as in a greater mean
residue ellipticity at 190 nm for the OGlcNAcylated peptide.
Data on the OGlcNAc and diethylphosphate peptides were
similar to those of a polyproline helix negative control, with the
threonines replaced by the sterically demanding tert-leuci-
ne.7,15a
and phosphorylation result in similar functional effects in
proteins.2
In order to identify whether the structural effects of post-
translational modifications seen in smaller peptides were also
observed in a broader structural context, we examined the
peptide tau211−238. This peptide contains six phosphorylation
sites, incorporating two proline-rich regions (residues 211−219
and 229−238) separated by a hydrophobic segment (the “B”
domain of Figure 2, including the highly hydrophobic VAVV
motif). By circular dichroism, phosphorylation of tau211−238
induced an increase in mean residue ellipticity at 228 nm,
consistent with induced polyproline helix upon tau phosphor-
ylation in data seen in smaller peptides above (Figure 5). The
Similar opposing effects of phosphorylation versus OGlcNA-
cylation were also observed in other proline-rich peptides
exhibiting residual PPII structure (tau211−219, tau229−238), with
phosphorylation inducing PPII and OGlcNAcylation opposing
PPII (Figure 4a,b). These peptides include the sites of several
major tau phosphoepitopes (pThr212, pSer214, and pThr231)
that are observed pathologically in Alzheimer’s disease. The
reduction in PPII for the OGlcNAcylated and diethylphos-
phorylated peptides, particularly compared to the phosphory-
lated peptides, was observable in reduced mean residue
ellipticity at 228 nm, in a red shift in the minimum around
200 nm, and in increased mean residue ellipticity at 190 nm.
In contrast, the structural effects of OGlcNAcylation and
phosphorylation were not distinct in tau196−209, although,
interestingly, peptides with both post-translational modifica-
tions were different from the unmodified peptide (Figure 4c).
In tau196−209, both OGlcNAcylation and phosphorylation
disrupted a nascent α-helix CD signature (minimum in CD
∼220 nm) in the unmodified peptides. This sequence is less
proline-rich than the other peptides examined (3 Pro in 14
residues), and includes three consecutive PG(S/T) repeats.
PGSPG(S/T) sequences in the PDB are observed as α-helix
nucleation sites, with the Ser side chain and SPG(S/T) main
chain oxygens acting as hydrogen bond acceptors to nucleate
(N-cap) the N-terminus of an α-helix (e.g., glutaminyl cyclase
(pdb 3si0), interleukin-5 receptor (pdb 3qt2)).20 Phosphor-
ylation has been observed to disrupt α-helix formation when
the phosphorylation site is at an internal site in α-helices.21 In
contrast to the results above, in this case, the diethylphosphate,
which induced increased α-helix, was structurally divergent
from OGlcNAcylation. The observation here of similar effects
of phosphorylation and OGlcNAcylation on α-helicity
emphasizes the importance of structural context in under-
standing the effects of phosphorylation and OGlcNAcylation.
Indeed, while in many cases phosphorylation and OGlcNAcy-
lation are functionally opposing, in some cases OGlcNAcylation
Figure 5. CD spectra of unmodified (green squares) and
phosphorylated (red circles) tau211−238 at 0.5 °C in water with 5
mM phosphate buffer pH 8 and 25 mM KF.
magnitude of the increase in mean residue ellipticity at 228 nm
was substantially less than seen in smaller peptides, and cannot
be definitively structurally assigned in the larger peptide, but is
consistent with an equivalent change in structure to polyproline
helix within the proline-rich segments. The magnitude of the
change in mean residue ellipticity is smaller because of the
larger number of residues in the peptide, including residues not
affected by the local structural organization induced by
phosphorylation (signal dilution by other residues in the
peptide, including B domain residues that adopt an extended
conformation in tau18). The data from these peptides confirm
induced polyproline helix upon phosphorylation in larger
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peptides but emphasize the difficulty in identifying polyproline
helix in larger peptides and the special utility of smaller peptides
for definitively identifying polyproline helix.
consistent with analysis of Griesinger, Mandelkow, Zweck-
stetter, and co-workers, who found that residues 240−251 of
nonphosphorylated tau do not adopt a well-defined con-
formation,5e,18,25 and that residues 232−239 predominantly
adopt a polyproline helix conformation, as we observed above
and previously7 for tau229−238 and tau229−242. Phosphorylation of
Ser235 resulted in a small increase in α-helicity of this peptide,
consistent with the results of Landrieu and Lippens, with a
greater α-helical induction observed in the α-helix-promoting
solvent TFE.24 In contrast, phosphorylation at both Ser235 and
Ser237, in addition to exhibiting a weak α-helical signature,
induced a small positive band at ∼225 nm consistent with the
local induction of polyproline helix around these residues that
was seen in tau229−238. Overall, these results are consistent with
the expected low α-helicity of this peptide sequence, whose α-
helicity is hampered by a short sequence of potential α-helical
character (12 residues; Pro has good α-helical propensity only
at the first and second residues of an α-helix26), multiple
residues with low α-helix propensity (3 Ser and a Thr within
the central 10 residues between the prolines), and a C-terminal
proline residue, which prevents continuation of the hydrogen-
bonding pattern of the α-helix and substantially reduces α-
helical content of short α-helical peptides.23b,d,27 Notably, the
α-helical content and induced α-helicity could be substantially
greater in the dynamic presence of transient tertiary structure
present in tau.4,18,28
One additional peptide, tau234−251 (Figure 2), was examined
by circular dichroism. Landrieu and Lippens identified via 13Cα
chemical shift index22 analysis that enzymatic phosphorylation
of a tau protein fragment (residues 208−324) by cdk2/
cyclinA3, including phosphorylation at Ser235, resulted in an
increase in 13Cα chemical shift consistent with a small
induction of α-helix in residues 236−239 of tau.5e Analysis of
the tau sequence suggests a short segment between residues
235 and 246 with the potential to form an α-helix, with the
sequence bounded by prolines at residues 236 and 247 serving
as α-helix start and stop signals.23 The C-terminal prolines at
residues 247, 249, and 251 are expected to strongly prevent α-
helix propagation beyond residue 246, although the
P
247VPMP251 sequence could potentially function as a hydro-
phobic α-helix C-cap. CD experiments revealed a very weak α-
helical signature in the nonphosphorylated peptide, with
modestly increased α-helicity in trifluoroethanol (TFE) (Figure
6, Figure S6, Supporting Information).24 These data are
To understand the structural basis for the observed opposing
conformational effects of OGlcNAcylation versus phosphor-
ylation in proline-rich motifs, all tau peptides were examined by
NMR spectroscopy. In addition to analysis of tau peptides, the
effects of post-translational modifications were examined within
the simple tau174−183-derived model peptide Ac-KXPP-NH2 (X
= Ser, Thr, or phosphorylated or OGlcNAcylated Ser or Thr),
whose sequence (with Thr) is repeated twice in tau174−183
(K174TPP177 and K180TPP183) and which is homologous to the
R230TPP233 sequence in tau229−238. This peptide was also
applied to examine the effects of threonine versus serine
modification. In addition, the structural effects of phosphor-
ylation versus OGlcNAcylation were also examined within the
model peptide Ac-GPPXPPGY-NH2 context, which was
previously used to identify polyproline helix propensity,15a
and in the related proline-rich peptide Ac-GPKXPPGY-NH2,
which contains the KTPP sequence present in tau174−183 (for
Figure 6. CD spectra of unmodified (green squares), mono-
phosphorylated at Ser235 (magenta open circles), and doubly
phosphorylated (at Ser235/Ser237) (red circles) tau234−251 at 25 °C
in water with 5 mM phosphate buffer pH 8 and 25 mM KF.
Figure 7. CD spectra of (a) Ac-KTPP-NH2 and (b) Ac-KSPP-NH2 peptides at 25 °C in water with 5 mM phosphate pH 8 and 25 mM KF.
Unmodified Ser/Thr, green squares; phosphoserine/phosphothreonine, red circles; Ser/Thr OGlcNAc, blue diamonds; Ser/Thr OPO3Et2, black
triangles. Data on these peptides at 2 °C, where greater PPII is observed, are in the Supporting Information (Figure S11).
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Figure 8. 1-D NMR spectra (amide region) of peptides with Ser/Thr, Ser/Thr(OGlcNAc), Ser/Thr(OPO3H−) (pH 4), Ser/Thr(OPO3(H)−/2−
)
(pH 6.5), and Ser/Thr(OPO32−) (pH 8). Experiments were conducted at 298 K in 90% H2O/10% D2O with 5 mM phosphate (pH 4 or as
indicated) and 25 mM NaCl. gS and gT indicate the resonances of the SerOGlcNAc and ThrOGlcNAc, respectively, backbone amide protons.
GlcNAc(NHAc) indicates the sugar amide proton. (a) tau174−183 peptides; (b) tau211−219 peptides; (c) tau229−238 peptides; (d) Ac-KTPP-NH2
peptides.
a
Table 1. Ser/Thr NMR Data at 298 K for Peptides
3JαN, Hz
ROPO3H−
ROPO3
ROGlcNAc
ROPO3Et2
2−
peptide
residue
ROH
tau174−183
tau211−219
Thr
Thr
Ser
7.3, 7.3
7.0, 6.4
6.5
6.7, 6.3
6.7, 6.6
n.d.
3.8, 3.5
3.7, 3.1
5.5
7.5, 7.5
5.7, 4.2
7.8
n.d.
7.0, 6.8
7.3
tau229−238
Thr
Ser
7.3
7.1
3.7
6.5
5.9
n.d.
7.3
6.4, 6.4
6.5
5.3, 5.2
3.5
8.0, 6.9
5.9
7.4, 7.0
7.9
GPPTPPGY
GPKTPPGY
KTPP
Thr
Thr
Thr
Ser
7.4
6.7
3.5
6.7
8.3
7.2
6.1
3.5
6.4
8.4
KSPP
6.8
6.5
5.5
6.4
7.6
mean
Ser
6.8
6.4
5.4
7.3
7.3
Thr
7.2
6.6
3.5
6.3
7.4
a
3JαN values < 6 Hz correlate with compact and ordered conformations, values > 8 Hz indicate extended conformations, and values between 6 and 8
Hz are observed in disordered peptides. In general, smaller 3JαN values indicate more compact conformations, while larger 3JαN values indicate more
extended conformations. tau196−209 is not included here because of spectral overlap. n.d. = not determined due to spectral overlap. Glu 3JαN values in
3
tau174−183(Thr → Glu) are 6.6 and 5.8 Hz. tert-Leucine JαN values in tau174−183(Thr → Tle) are 8.8 and 7.9 Hz. In Ac-GPPXPPGY-NH2 peptides,
the 3JαN value of Glu is 6.3 Hz, the third most-restricted value for canonical amino acids after Ala (5.7 Hz) and Asp (6.2 Hz), while that of tert-leucine
is 8.3 Hz, larger than all canonical amino acids (Val 8.0 Hz, Ile 7.9 Hz) and indicative of a strong preference for the extended conformation.15a Data
on phosphorylated peptides were obtained at pH 4 (ROPO3H−) or pH 8 (ROPO32−) (typical phosphoserine/phosphothreonine pKa 5.5−6.0).
Additional NMR data for all peptides are in the Supporting Information.
these peptides, X = ThrOH, ThrOPO32−, ThrOPO3Et2, and
ThrOGlcNAc).
In these proline-rich model peptides, similar conformational
effects of post-translational modifications were observed by CD
as were found in proline-rich tau peptides, with phosphor-
ylation increasing PPII and OGlcNAcylation and diethylphos-
phorylation opposing PPII (Figure 7, Figures S7−S17, Tables
S5−S7, Supporting Information). Notably, comparison of the
CD spectra of KTPP and KSPP peptides (Figure 7, Figures
S7−S11, Tables S5 and S6, Supporting Information) revealed a
3808
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Figure 9. 1H−15N HSQC spectra of (a) tau174−183, (b) tau211−219, (c) tau229−238, and (d) Ac-KTPP-NH2 peptides. Green, peptides with unmodified
Ser/Thr; blue, peptides with Thr(OGlcNAc); magenta, peptides with Ser/Thr(OPO3H−) (pH 4); red, peptides with Ser/Thr(OPO32−) (pH 8).
substantially larger structural change for Thr phosphorylation
than Ser phosphorylation (Δ[θ]224 = +5310 and +2230 deg
cm2 dmol−1 for pThr−Thr and pSer−Ser, respectively). The
larger change in structure upon Thr phosphorylation than Ser
phosphorylation was both due to lower population of PPII for
the peptide with Thr than with Ser and due to greater PPII with
pThr than with pSer. These data suggest that substantially
larger structural changes are induced because of threonine
phosphorylation than because of serine phosphorylation.
Data on polyproline helix model peptides Ac-GPPTPPGY-
NH2 and Ac-GPKTPPGY-NH2 peptides were similarly
consistent with data in tau peptides. Data from Ac-
GPPpTPPGY-NH2 also indicated no effect of 2 mM MgCl2
on the CD spectra (and thus, no substantial effect of Mg2+ on
structure) of phosphorylated peptides. In addition, a greater
mean residue ellipticity at 228 nm was observed at 2 °C than at
25 °C, consistent with the interpretation that these CD data are
specifically indicative of PPII content in the peptides, as was
previously seen in other proline-rich and polyproline helix-
containing peptides.7,15a−c,16a,e
A series of homonuclear (1-D 1H and TOCSY) and
1
1
heteronuclear (1H−15N HSQC, H−13C HSQC, and H−13C
HMBC) NMR experiments was conducted on tau-derived
peptides and proline-rich model peptides to identify residue-
specific and post-translational-modification-specific changes in
structure (Figures 8−11, Tables 1−4). NMR data in this series
of peptides were consistent with CD data, indicating that
phosphorylation in proline-rich sequences induces structural
changes leading to more compact and more ordered
conformations, whereas OGlcNAcylation and diethylphosphor-
ylation exhibited evidence of more extended conformations, as
expected for sterically demanding amino acids in proline-rich
domains.7,15a−c,16e 1-D 1H NMR spectroscopy indicated
substantial divergence of the peptides that was a function of
post-translational modification: across all peptides, relative to
unmodified Ser/Thr, phosphorylation induced downfield amide
chemical shifts and smaller 3JαN for phosphorylated residues; in
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1
Table 2. Mean H Chemical Shifts (ppm) for Serine/Threonine Resonances Across All Peptides as a Function of Side Chain
a
and Post-Translational Modification
HN
Hα
Hβ
OPO3H−
OPO3
OH
OGlcNAc
OPO3H−
OPO3
OH
OGlcNAc
OPO3H−
OPO3
2−
2−
2−
OH
OGlcNAc
Ser
8.32
8.23
8.31
8.01
8.62
8.43
8.99
9.63
4.50
4.56
4.50
4.55
4.68
4.63
4.63
4.35
3.87
4.11
3.79
4.12
4.13
4.43
4.05
4.27
Thr
a
Full tabulated NMR data and statistical analysis are in the Supporting Information.
Table 3. Mean Amide 15N Chemical Shifts (ppm) for Serine/
Threonine Resonances Across All Peptides as a Function of
Side Chain and Post-Translational Modification
contrast, OGlcNAcylation of Thr residues induced upfield
amide chemical shifts and amide chemical shifts similar to those
of Ser for SerOGlcNAc. In addition, experiments on non-
phosphorylated and phosphorylated tau211−238 indicated that
the large amide chemical shift changes and conformational
restriction observed for phosphoresidues in tau211−219 and
tau229−238 peptides were also observed in the larger peptide
context (Figures S54 and S55, Supporting Information). Of
particular note, in all peptides, phosphorylated residues
exhibited substantially downfield (δ = 9.4−9.8 ppm as
ThrOPO32−, 8.7−9.2 ppm as SerOPO32−) amide proton
chemical shifts, with amide proton chemical shifts substantially
more downfield for the dianionic than the monoanionic
phosphates (monoanionic pSer/pThr δ = 8.35−8.75 ppm)
(Figures 8 and 9, Table 2, Table S36, Supporting Information),
as has been observed previously in some peptides and
proteins.5a,c,e,29
a
b
2−
OH
OPO3H−
OPO3
Δδ
Ser
118.2
118.1
116.9
119.3
120.8
124.3
2.6
6.2
Thr
a
Full tabulated NMR data and statistical analysis are in the Supporting
b
Information. Δδ = δ (Ser/Thr(OPO32−)) − δ (Ser/Thr(OH)).
threonine exhibited amide nitrogen chemical shifts only 0.6
ppm downfield of those of threonine. The large divergence in
amide nitrogen chemical shift between monoanionic and
dianionic phosphothreonine is not consistent with differences
in the electron-withdrawing nature of the different protonation
states of these phosphates,13b,32 suggesting that these differ-
ences are due to particular structure induced by dianionic
Phosphorylated Ser/Thr residues in these peptides were
3
3
particularly conformationally restricted. JαN values correlate
phosphothreonine, consistent with differences in JαN values
between monoanionic and dianionic phosphopeptides. 1H−15N
with the ϕ backbone torsion angle via a parametrized Karplus
equation, with values between 6 and 8 Hz consistent with
disorder or averaging of multiple conformations, values > 8 Hz
indicative of ordered, extended conformations, and values < 6
Hz indicative of ordered, compact conformations, and more
broadly with smaller values indicating more compact
conformations, larger values indicating more extended con-
formations, and values further from random coil values
HSQC data from other proline-rich peptides (tau211−219
,
tau229−238, and Ac-KTPP-NH2) exhibited similar trends, with
large downfield changes in amide hydrogen and amide nitrogen
resonances for dianionic phosphoresidues compared to the
monoanionic phosphoresidues or unmodified Ser/Thr [amide
nitrogens: tau211−219 Δδmean = +0.1 and +1.0 ppm for
monoanionic phosphoserine and phosphothreonine, respec-
tively, compared to unmodified Ser/Thr, versus Δδmean = +2.9
and +5.5 ppm for dianionic phosphoserine and phosphothreo-
nine, respectively, compared to unmodified Ser/Thr; tau229−238
Δδmean = +2.6 ppm (pSer(OPO32−)) and +5.6 ppm (Thr-
(OPO32−)) ppm for the dianionic phosphoresidues compared
to the unmodified Ser/Thr; Ac-KTPP-NH2 Δδ = +1.1 ppm
(pThr(OPO3H−)) and Δδ = +6.5 ppm (pThr(OPO32−))
relative to unmodified Thr; Ac-GPPTPPGY-NH2, Δδ = +0.1
ppm (pThr(OPO3H−)) and Δδ = +6.2 ppm (pThr(OPO32−))
relative to unmodified Thr]. Collectively, these data demon-
strate very large induced changes in the electronic environment
around the amide nitrogens of dianionic phosphoserine and
dianionic phosphothreonine residues compared to Ser/Thr,
Ser/Thr(OGlcNAc), or to monoanionic phosphoserine or
phosphothreonine.
Notably, small downfield changes in amide nitrogen chemical
shift are associated with the polyproline helix conformation
(Δδ = +1.1 ppm for change from random coil to polyproline
helix).16e While the changes in phosphoserine/phosphothreo-
nine amide chemical shifts are too large to be explained by
secondary structure, most other resonances in these peptides
also exhibited small downfield changes in 15N amide chemical
shift upon phosphorylation, consistent with the increased PPII
seen by CD (Figure 10; tabulated data in the Supporting
Information). In contrast, the amides of OGlcNAcylated
tau174−183 exhibited small upfield changes in 15N chemical
shift, consistent with the reduced PPII seen in this peptide.
indicating greater extent of order.30 The JαN values observed
3
are indicative of special conformational order for the
phosphorylated residues: across all peptides, dianionic
3
phosphothreonine exhibits a mean JαN = 3.5 Hz, correspond-
ing to ϕ = −55°, compared to a random coil value for Thr (3JαN
= 7.2 Hz, average ϕ = −83°); dianionic phosphoserine exhibits
3
a mean JαN = 5.4 Hz, corresponding to average ϕ = −70°,
compared to a random coil value for Ser (3JαN = 6.8 Hz, average
ϕ = −80°)) (Table 1).30 Notably, the substantial conforma-
tional order induced by phosphorylation was dependent on the
dianionic phosphates: only small increases in order were
induced by monoanionic phosphoserine (3JαN = 6.4 Hz),
phosphothreonine (3JαN = 6.6 Hz), and glutamic acid (3JαN
=
6.2 Hz). Interestingly, the small coupling constants for
dianionic phosphorylated amino acids observed herein are
also consistent with a growing number of examples of proteins
in which phosphorylation induces α-helix formation when at its
N-terminus.5e,21b,29a,31
1H−15N HSQC experiments indicated that, in addition to
large downfield changes in the chemical shifts of amide protons,
the serine/threonine amide nitrogens exhibited large downfield
changes in chemical shift upon phosphorylation across all
peptides (Figure 9, Table 3; tabulated data Table S39,
Supporting Information). In tau174−183, the dianionic phospho-
threonine amide nitrogens were 7.2 ppm downfield of the
amides of threonine and 6.6 ppm downfield of the amides of
monoanionic phosphothreonine. In contrast, OGlcNAcylated
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1
Figure 10. (a−d) H−13C HSQC spectra (Hα−Cα region) of (a) tau174−183, (b) tau211−219, (c) tau229−238, and (d) Ac-KTPP-NH2 peptides. (e,f)
1H−13C HMBC spectra (HαCO region) of (e) tau174−183 and (f) Ac-KTPP-NH2 peptides. Green, peptides with unmodified Ser/Thr; red,
peptides with Ser/Thr(OPO32−) (pH 8). Full spectra and tabulated data are in the Supporting Information.
1H−13C HSQC experiments were conducted on tau peptides
to further characterize the residue-specific effects of protein
phosphorylation (Figure 10, Table 4). In addition, to determine
the effects of phosphorylation on the backbone carbonyls,
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Table 4. Mean 13C Chemical Shifts (ppm) for Serine/
Threonine Resonances Across All Peptides as a Function of
a
Side Chain and Post-Translational Modification
13Cα
13Cβ
b
2−
2−
OH
OPO3
Δδ
OH
OPO3
Δδ
Ser
53.3
57.1
53.0
58.4
−0.3
1.3
60.7
66.9
62.6
70.0
1.9
3.1
Thr
a
Full tabulated NMR data and statistical analysis are in the Supporting
b
Information. Δδ = δ (Ser/Thr(OPO32−)) − δ (Ser/Thr(OH)).
1H−13C HMBC experiments were conducted on nonphos-
phorylated and phosphorylated tau174−183 and the model
peptide Ac-KTPP-NH2 (Figure 10e,f). PPII, in contrast to α-
helix or β-sheet, does not exhibit large changes in Hα (Δδ =
−0.03 ppm), Cα (Δδ = +0.3 ppm), or CO (Δδ = +0.1 ppm)
chemical shift compared to random coil.16e These experiments
1
revealed large changes in the H and 13C chemical shifts of
phosphorylated residues, as expected because of the electronic
change of the side chain, though with substantially larger
chemical shift changes at phosphothreonine than at phospho-
serine (Table 3). In particular, as the dianion, phosphorylation
of serine induced downfield shifts in Hα (Δδ = +0.13 ppm)
and upfield shifts in Cα (Δδ = −0.3 ppm), in contrast to
upfield shifts in Hα (Δδ = −0.21 ppm) and downfield shifts in
Cα (Δδ = +1.3 ppm) for phosphorylation of threonine. Data
from other residues, within the context of the small inherent
changes in chemical shift for PPII, also indicated that the
structural changes upon phosphorylation propagated to
residues beyond the phosphorylated residues (Figure 10).33
One defining feature stabilizing the polyproline helix is an n →
π* interaction between adjacent carbonyls.34 The observation
of changes in the carbonyl chemical shifts across all residues
(including the N-terminal acetyls) is consistent with phosphor-
ylation-induced changes in the environment around the
backbone carbonyls of all residues in the peptides.
Figure 11. (a) Temperature-dependent 1H NMR spectra of Ac-
KT(OPO32−)PP-NH2. Experiments were conducted in 90% H2O/10%
D2O with 5 mM phosphate buffer and 25 mM NaCl at pH 8.0.
Experiments were conducted at 277 (top), 298, 308, 323, and 338 K
(bottom). Experiments at 277, 298, and 308 K were conducted on a
600 MHz cryoprobe instrument, while experiments at 323 and 338 K
1
were conducted on a 400 MHz instrument. (b) Salt-dependent H
NMR spectra of Ac-KT(OPO32−)PP-NH2. Experiments were
conducted in 90% H2O/10% D2O in 5 mM phosphate buffer at pH
8.0 with 25 (top), 125, 225, 325, and 1000 (bottom) mM NaCl.
Interestingly, by NMR, the effects of modifications on serine
versus threonine were divergent in magnitude (Tables 1−4), as
had been seen by CD above in Ac-KXPP-NH2 peptides (Figure
5). Greater overall conformational restriction (3JαN) was
observed at phosphothreonine than at phosphoserine, as well
Peptides with phosphorylated amino acids also exhibited
relatively slow amide hydrogen exchange considering the
absence of tertiary structure or hydrogen-bonded secondary
structure, with most amide protons observable at pH 8 for
phosphorylated proline-rich peptides (Figure 8, Supporting
Information). For the peptide Ac-KT(OPO32−)PP-NH2, slow
3
as a larger change in JαN (and thus in the main chain torsion
angle ϕ) between the nonphosphorylated and phosphorylated
peptides (mean Δ3JαN = 3.7 Hz for Thr, versus 1.4 Hz for Ser).
Phosphothreonine amide protons also exhibited greater
downfield shifts than phosphoserine amides (mean amide δ
9.63 ppm for dianionic pThr, versus 8.99 ppm for dianionic
pSer, compared to 8.23 and 8.32 ppm for Thr and Ser; Δδmean
3
amide exchange, small JαN values, and downfield pThr amide
chemical shifts at pH 8 were persistent even at elevated
temperature (up to 323 K) and at high salt concentrations (up
to 1 M NaCl) (Figure 11). These data are consistent with a
strong interaction that is in slow exchange and that cannot be
readily screened electrostatically, suggesting that the induced
structure is not primarily due to a simple lysine-phosphate or
arginine-phosphate electrostatic interaction of the phosphory-
lated residue with the prior basic residue. These data on
dianionic phosphorylated amino acids are particularly striking,
since in disordered peptides, amide protons are generally not
observed or are substantially exchange-broadened at pH 8. Slow
amide exchange on the NMR time scale at pH 8 is generally
associated with more stable structures (e.g., those involving
hydrogen bonding, an interaction that could slow amide
exchange at the pSer/pThr amides, though not other
amides).29d,e,35 While PPII does not exhibit hydrogen bonding,
PPII is stabilized by n → π* interactions between adjacent
carbonyls, which could also potentially slow exchange of the
amide protons.34a−d,36
= +1.40 ppm for threonine phosphorylation, versus Δδmean
=
+0.67 ppm for serine phosphorylation (Table 2)), as well as
greater downfield shifts in amide nitrogens (mean amide δ
124.3 ppm for dianionic pThr, versus 120.8 ppm for dianionic
pSer, compared to 118.1 and 118.2 ppm for Thr (Δδmean = +6.2
ppm) and Ser (Δδmean = +2.6 ppm), respectively) (Table 3).
Larger changes in chemical shifts were also observed for
threonine phosphorylation than serine phosphorylation on Hα,
Cα, and Cβ (Table 2, Table 4, Tables S37 and S40, Supporting
Information). Collectively, these data are consistent with a
stronger phosphate-amide interaction in phosphothreonine
than phosphoserine and greater induced structural changes
for phosphothreonine than phosphoserine. Notably, threonine
and serine residues are differentially phosphorylated and
dephosphorylated in vivo, and evolution of Thr and Ser
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residues occurs at different rates, suggesting native functional
differences between Thr and Ser.37
OGlcNAcylation are somewhat similar to the free Ser/Thr
hydroxyls and specifically oppose the effects of phosphor-
ylation, consistent with the observed effects of OGlcNAcylation
in opposing tau aggregation.
DISCUSSION
■
Phosphorylation of threonine residues was found herein to
induce greater structural changes than serine phosphorylation,
although both phosphorylation events induced more ordered
conformations, including induced PPII, restriction of ϕ, and
slower amide exchange.21a,31c,43 The greater structural change
at Thr was due to a combination of both more disorder for
nonphosphorylated Thr than Ser and more induced order for
pThr than pSer, with particular conformational restriction of ϕ
and evidence consistent with a phosphate-amide hydrogen
bond. To identify a possible structural basis for these
observations, phosphothreonine residues in several high
resolution crystal structures (protein−protein interactions
(see below) and globular proteins) were analyzed. The
crystallographic data revealed a significant degree of conforma-
tional restriction in some phosphothreonine residues, con-
sistent with the data herein. The conformation of phospho-
threonine 197 in protein kinase A (pdb 1rdq, the highest
resolution (1.26 Å) protein with pThr in the PDB) exhibits
pThr in a PPII conformation (ϕ = −67°, ψ = +134°), χ1 =
−53° (g−), χ2 = +119° (surprisingly, eclipsing C−H/O−P
bonds), a hydrogen bond between the phosphate and the
phosphothreonine amide, and close interaction between the n
− 1 carbonyl (conjugated to the hydrogen-bonded pThr
amide) and the pThr carbonyl (O−C distance 2.88 Å,
substantially less than the 3.22 Å sum of van der Waals radii,
We have described the direct comparison of the structural
effects of phosphorylation versus OGlcNAcylation, competing
intracellular protein post-translational modifications that often
occur on the same Ser/Thr residues, on peptide conformation
within a typical natively disordered protein context.2,18,38 This
work was specifically applied within the context of the tau
protein, where hyperphosphorylation is associated with protein
misfolding and aggregation, but OGlcNAcylation is protective
against protein misfolding.3a,5d,8−10 We found that within
proline-rich sequences phosphorylation promotes conforma-
tional order and PPII formation, whereas OGlcNAcylation or
modification with the practical OGlcNAc mimic of dieth-
ylphosphorylation leads to conformational preference against
PPII and a more disordered or extended conformation. The
effects of phosphorylation and OGlcNAcylation are divergent:
whereas phosphorylation induces significant conformational
order, particularly on threonine residues, the effects of
OGlcNAcylation are more subtle, confirming the strong
conformational biases of serine and threonine against PPII.
Interestingly, in the RNA polymerase II C-terminal domain
repeat (SYSPTSPS), phosphorylation promotes binding to the
Pin1 WW domain as a polyproline helix, whereas Thr
OGlcNAcylation induces a more extended conformation,
consistent with results on tau peptides herein.19 As
phosphorylation and OGlcNAcylation are competing protein
intracellular post-translational modifications, which generally
occur on disordered protein sequences, these data suggest
potentially general opposing modes of structural changes due to
these post-translational modifications.39
as well as an O−C−O angle of 107°, similar to the Burgi−
̈
Dunitz trajectory), as would be expected by a PPII-favoring n
→ π* interaction (Figure 12).44 Notably, Thr197 is a critical
Hyperphosphorylation of tau is associated with conforma-
tional changes leading to tau misfolding and aggregation as the
neurofibrillary tangles of Alzheimer’s disease.3,10 Aggregation of
tau is mediated by the hydrophobic tubulin-binding domains
(TBDs). The TBDs may be stabilized against aggregation by
binding to microtubules, or via association with the N-terminus
and C-terminus in a global hairpin conformation that masks the
hydrophobic residues of the TBDs.4,40 Phosphorylation or
pseudophosphorylation induces structural changes that open
this hairpin conformation, exposing the hydrophobic TBDs to
promote aggregation, while OGlcNAcylation is protective
against aggregation, potentially due to maintaining or stabilizing
the global hairpin, though to date no structural data exist to
suggest this latter possibility.41 The importance of the N-
terminus in protecting the TBDs from aggregation is
emphasized by the ability of N-terminal tau peptides to inhibit
tau aggregation, consistent with a critical role of the structure of
the proline-rich linker between these domains in maintaining
soluble tau.42 Thus, changes in secondary structure in tau’s
proline-rich domain may mediate global changes in tau
structure and function. Notably, phosphorylation at Ser-Pro
and Thr-Pro sites within the proline-rich domain inhibits the
ability of tau to promote tubulin polymerization.5e The most
striking data herein are the high degree of order observed at
Figure 12. Structure of phosphothreonine residue 197 in protein
kinase A (pdb 1rdq, 1.26 Å resolution).44 A similar conformation of
phosphothreonine (ϕ,ψ = −64°, +131°; χ1 = g−; eclipsing C−H/O−P
bonds (χ2 = +118°); phosphate-amide hydrogen bond; n → π*
interaction) was observed in a tau peptide containing pThr231 bound
to a monoclonal antibody (pdb 4glr), or of a pSer phosphopeptide
bound to Pin1 (pdb 1f8a).19b,46b Hydrogens were added in Pymol.
and conserved residue in the activation loop of PKA and related
protein kinases. Thr197 phosphorylation induces a substantial
disorder-to-order transition in PKA and increase in PKA
activity and stability, with Thr197 exhibiting no electron density
in nonphosphorylated PKA.45 The side chain conformational
restriction observed crystallographically in phosphothreonine
residues was also observed by NMR in Ac-KT(OPO32−)PP-
3
phosphothreonine residues, with JαN values that indicate very
stable non-random coil conformation at these residues. Overall,
these data indicate that phosphorylation of the tau proline-rich
domain induces significant conformational changes that result
in ordering of the proline-rich domain, particularly as observed
by NMR in the dianionic state, whereas the effects of
3
NH2, which exhibited JHαHβ = 9.5 Hz for phosphothreonine,
near the maximum of the Karplus curve and indicating χ1 ∼
−60° (g− rotamer) (compared to nonphosphorylated Thr
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3JHαHβ = 6.4 Hz, as expected for disorder; Figure S66,
adopted ordered structures, including induction of polyproline
helix. We found particular conformational restriction in
phosphothreonine residues, with a highly ordered structure
adopted. Notably, phosphoproteomics experiments have
revealed that over 25% of phosphorylation sites are at Ser-
Pro or Thr-Pro sequences, suggesting that the results observed
herein in tau peptides and in proline-rich model peptides may
have broad applicability in understanding the effects of
phosphorylation on protein structure, particularly in regions
of protein disorder.37a
3
Supporting Information), as well as JHβP = 9 Hz, which is
close to ideal for an eclipsing χ2 C−H/O−P bond (3JHβP = 9
Hz was also seen in tau174−183; see Figures S20 and S77,
Supporting Information). Collectively, the data observed herein
across multiple phosphothreonine-containing peptides, includ-
3
ing induction of polyproline helix by CD, small JαN (=
restricted ϕ) for phosphothreonine, large downfield changes in
1
amide H and 15N chemical shifts, and slow amide hydrogen
exchange, are consistent with this crystallographically observed
conformation of phosphothreonine, suggesting a potentially
general mode for conformational restriction by phosphothreo-
nine residues in both disordered and globular proteins.
EXPERIMENTAL SECTION
■
Peptide Synthesis and Characterization. Peptides were
synthesized by Fmoc solid phase peptide synthesis. All peptides
were acetylated at the N-terminus and contained C-terminal amides.
Complete synthetic procedures and characterization data are in the
Supporting Information.
We previously observed that nature often employs the
polyproline helix for phosphoprotein recognition by phospho-
serine/phosphothreonine-binding domains, including binding
of phosphopeptides in a polyproline helix by WW, FHA, Polo-
box, and BRCT domains, though not by 14−3−3 domains.7 In
addition, recent analysis of phosphorylation sites in intrinsically
disordered proteins suggests a greater propensity for PPII
around phosphorylation sites.37c Serine and threonine have
natively low PPII propensities, suggesting the possibility of
phosphorylation-mediated switches to PPII.7,15a In proline-rich
sequences, eukaryotic proteins also exhibit low frequencies of
Asp and Glu residues, but high frequencies of Ser and Thr
residues.15a These data suggest that phosphorylation can
provide both conformational preference for PPII and anion
specificity via electrostatics to protein-binding domains that
recognize phosphorylated proteins via a polyproline helix
conformation. Notably, tau phosphorylated at residue 231 is
observed in a polyproline helix both when bound to the Pin1
WW domain (pdb 1i8h) and to an antiphosphotau antibody
(pdb 4glr).46 In this case, antibody recognition, which involves
no inherent conformational bias in recognition epitopes,
utilized PPII for high affinity (Kd = 1.1 nM) and high
specificity (>500-fold specificity) recognition of the phosphory-
lated peptide over the nonphosphorylated peptide. In sum,
these data suggest that polyproline helix provides the possibility
of increased specificity and affinity in folding and recognition of
phosphorylated Ser/Thr, over both nonphosphorylated or
OGlcNAcylated Ser/Thr.
Circular Dichroism. CD spectra were collected on a Jasco J-810
Spectropolarimeter in a 1 mm cell at 25 °C. Peptide concentrations
were 15−400 μM in water containing 5 mM phosphate buffer (pH 8.0
or as indicated) and 25 mM KF. Data represent the average of at least
three independent trials. Data were background corrected but were
not smoothed. Error bars indicate standard error.
NMR Spectroscopy. NMR spectra of peptides were collected at
298 K on a Bruker AVC 600 MHz NMR spectrometer equipped with
̈
a triple resonance cryoprobe. 31P NMR spectra were recorded on a
Bruker DRX 400 MHz NMR spectrometer equipped with a BBO
̈
probe. Peptides were dissolved in buffer containing 5 mM phosphate
(pH 4.0, 6.5, 7.2, or 8.0) with 25 mM NaCl, 100 μM TSP, and 90%
3
3
H2O/10% D2O. JαN and JHP were determined directly from the 1-D
1H NMR spectra and proton-coupled 31P NMR spectra, respectively.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of protected Fmoc OGlcNAcylated amino acids,
peptide synthesis and characterization, CD data on model
peptides, and full NMR spectroscopy data. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
zondlo@udel.edu
■
Author Contributions
†M.A. Brister and A. K. Pandey contributed equally to this
work.
CONCLUSIONS
■
Phosphorylation and OGlcNAcylation are the most significant
intracellular post-translational modifications of serine and
threonine.2 We found opposing structural effects of phosphor-
ylation and OGlcNAcylation in proline-rich sequences, which
are among the most common sequences in eukaryotes,1 but
similar effects of both modifications in opposing the
unmodified hydroxyl in sequences with a nascent α-helix.
These data provide a plausible structural basis for the
observation that OGlcNAcylation of tau opposes neurofibrillary
tangle formation, because of its confirmation of the disordered
structure of sequences with unmodified serine and threonine
residues, while phosphorylation is associated with neuro-
fibrillary tangle formation, potentially because of a disorder to
order transition that promotes opening of the global hairpin
conformation of tau. More generally, these data provide a
context for interpreting sequence-specific structural effects of
these post-translational modifications, with broad potential
application to understanding the intracellular effects of
phosphorylation and OGlcNAcylation. Across all peptides,
dianionic phosphoserine and dianionic phosphothreonine
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Alzheimer’s Association (NIRG-06-14403), NIH
(GM93225), HHMI (undergraduate research fellowships for
M.A.B. and A.A.B.), and the University of Delaware for funding.
We thank Alaina Brown, Ben Liechty, and Krista Thomas for
preliminary structural studies on phosphorylated peptides.
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