Conformational changes associated with post-translational modifications of pro143 in Skp1 of dictyostelium -a dipeptide model system

Article abstract of DOI:10.1021/ja5033277

Prolyl hydroxylation and subsequent glycosylation of the E3^SCF ubiquitin ligase subunit Skp1 affects its conformation and its interaction with F-box proteins and, ultimately, O₂-sensing in the organism. Taking a reductionist approach to understand the molecular basis for these effects, a series of end-capped Thr-Pro dipeptides was synthesized, tracking the sequential post-translational modifications that occur in the protein. The conformation of the pyrrolidine ring in each compound was gauged via coupling constants (³J_Hα,Hβ) and the electronegativity of the Cγ-substituents by chemical shifts (¹³C). The equilibrium between the cis-trans conformations about the central prolyl peptide bond was investigated by integration of signals corresponding to the two species in the ¹H NMR spectra over a range of temperatures. These studies revealed an increasing preference for the trans-conformation in the order Pro < Hyp < [α-(1,4)GlcNAc]Hyp. Rates for the forward and reverse reactions, determined by magnetization transfer experiments, demonstrated a reduced rate for the trans-to-cis conversion and a significant increase in the cis-to-trans conversion upon hydroxylation of the proline residue in the dipeptide. NOE experiments suggest that the Thr side chain pushes the sugar away from the pyrrolidine ring. These effects, which depended on the presence of the N-terminal Thr residue, offer a mechanism to explain altered properties of the corresponding full-length proteins.

Full text of DOI:10.1021/ja5033277

Article
pubs.acs.org/JACS
Conformational Changes Associated with Post-Translational
Modifications of Pro143 in Skp1 of DictyosteliumA Dipeptide Model
System
Chamini V. Karunaratne,† Thomas K. Weldeghiorghis,† Christopher M. West,‡ and Carol M. Taylor*,†
†Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, United States
‡Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health
Sciences Center, Oklahoma City, Oklahoma 73104, United States
S
* Supporting Information
ABSTRACT: Prolyl hydroxylation and subsequent glycosylation of
the E3SCF ubiquitin ligase subunit Skp1 affects its conformation and
its interaction with F-box proteins and, ultimately, O2-sensing in the
organism. Taking a reductionist approach to understand the
molecular basis for these effects, a series of end-capped Thr-Pro
dipeptides was synthesized, tracking the sequential post-translational
modifications that occur in the protein. The conformation of the
pyrrolidine ring in each compound was gauged via coupling constants (3JHα,Hβ) and the electronegativity of the Cγ-substituents
by chemical shifts (13C). The equilibrium between the cis−trans conformations about the central prolyl peptide bond was
1
investigated by integration of signals corresponding to the two species in the H NMR spectra over a range of temperatures.
These studies revealed an increasing preference for the trans-conformation in the order Pro < Hyp < [α-(1,4)GlcNAc]Hyp. Rates
for the forward and reverse reactions, determined by magnetization transfer experiments, demonstrated a reduced rate for the
trans-to-cis conversion and a significant increase in the cis-to-trans conversion upon hydroxylation of the proline residue in the
dipeptide. NOE experiments suggest that the Thr side chain pushes the sugar away from the pyrrolidine ring. These effects,
which depended on the presence of the N-terminal Thr residue, offer a mechanism to explain altered properties of the
corresponding full-length proteins.
INTRODUCTION
■
The process whereby proteins are polyubiquitinated and
delivered to the 26S proteasome for degradation involves
several steps and is fundamental to the maintenance of healthy
levels of all proteins in eukaryotic organisms. A prominent
really interesting new gene (RING)-type third order (E3)
ubiquitin ligase, referred to as the Cullin-RING-Ligase-1
(CRL1) or the Skp1-Cullin-F-box (SCF) complex, consists of
several subunits including the S-phase kinase-associated protein
1 (Skp1).1 In Dictyostelium discoideum, the proline residue at
Figure 1. Glycosylated region of Skp1.
position 143 of Skp1 is hydroxylated by a cytoplasmic prolyl
hydroxylase (PhyA).2 We have recently confirmed that the
hydroxylation, by analogy to the hypoxia inducible factor α
αGal.10 Biophysical studies, viz. circular dichroism (CD) and
(HIFα) prolyl hydroxylases, delivers (2S,4R)-4-hydroxyproline
(Hyp).3 Hydroxylation of Skp1 is important for the proper
oxygen-dependence of differentiation of the organism into a
fruiting body to disperse spores.4,5 Polypeptide N-acetylgluco-
saminyl-transferase 1 (Gnt1) transfers N-acetylglucosamine
(GlcNAc) from uridine diphosphate (UDP) to Hyp143 of
Skp1.6,7 The installation of the first GlcNAc residue renders
Skp1 subject to further glycosylation by the PgtA (β3GalT/
α2FucT) and AgtA (αGalT) glycosyltransferases,8−10 which
modulate oxygen-dependence conferred by prolyl hydroxyla-
tion. The hypothesized terminal structure, illustrated in Figure
1, has been validated for all but the linkage of the terminal
small-angle X-ray scattering (SAXS), indicate that the αGlcNAc
residue promotes α-helical secondary structure and increased
order of the polypeptide.11 These post-translational modifica-
tions occur in other unicellular eukaryotes, including
Toxoplasma gondii.8,12,13
While a fundamental impact of glycosylation on protein
structure and function has been widely appreciated for many
years, the empirical basis for structural changes is less well
understood. Previous studies have focused on the more
Received: April 9, 2014
© XXXX American Chemical Society
A
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ubiquitous O-glycosidic linkages to serine and threonine
residues.14−18
Scheme 2. Dipeptides Synthesized
Prior to hydroxylation, the pyrrolidine ring of the Pro143
residue is likely to adopt a Cγ-endo conformation (Scheme 1).
Scheme 1. Pyrrolidine Conformation
X-ray crystallographic studies show that the Thr-Pro143 peptide
bond favors the trans conformation (ω = 180°) when the Skp1
proteins from Saccharomyces cerevisiae or Arabidopsis thaliana
are complexed to an F-box protein.19,20 This cis−trans
isomerism might be important in Skp1 folding, perhaps
mediated by a Thr-Pro prolyl isomerase.21 By analogy to
recent findings in the orthologous human HIFα,22,23 upon
hydroxylation by a trans-4-prolylhydroxylase, there could be
dramatic changes in the topology of this region of Skp1, viz.
pyrrolidine ring-flip to the Cγ-exo conformation and a
reinforced preference for a trans amide bond (ω = 180°),
which may in turn influence processing by Gnt1 and F-box
protein binding. With a GlcNAc residue installed, torsion
angles about the glycosidic bond will contribute to recognition
by other proteins, including F-box proteins and the β3GalT/
α2FucT that introduces the next two monosaccharides.12,24,25
These interrelated conformational effects may provide the
trigger for α-helix formation in Skp1.
compounds 2 and 3 were synthesized and used to confirm the
4R configuration of the hydroxproline residue in Skp1.3 In
addition, Ac-[α-(1,4)GlcNAc]Hyp-NHMe (5) was prepared.
This compound represents the GlcNAc analogue of Ac-[α-
(1,4)Gal]-Hyp-NHMe described previously by Schweizer and
co-workers.27 Full details of the synthesis of the compounds
depicted in Scheme 2 are provided in the Supporting
Information. In the case of glycosides 4 and 5, glycosylation
was performed with thioglycoside donor 6 using NIS/AgOTf
activation30,31 and an appropriate carbamate protected
hydroxyproline ester.
1
3
Analysis of the H NMR J coupling constants Jαβ1 and Jαβ2
(α, β etc. as defined for compound 1 in Scheme 2) gave an
indication of the conformational preference of the pyrrolidine
ring in each compound (Table 1). According to the literature,
Hydroxyproline glycosides are common in plants; however,
no examples have been reported to-date in mammalian
systems.26 In 2007, Schweizer and co-workers reported on
the thermodynamics and kinetics of cis−trans isomerization of a
family of proline derivatives, Ac-Pro*-NHMe where Pro* was
L-proline (Pro), trans-4-hydroxy-L-proline (Hyp), trans-4-tert-
butoxy-L-proline, α-(1,4)-galactosyl-trans-4-hydroxy-L-proline
[α-(1,4)-Gal-Hyp], and β-(1,4)-galactosyl-trans-4-hydroxy-L-
proline [β-(1,4-Gal)-Hyp].27 The surprising result of this
work was that, within experimental error, the equilibrium
constants and rate constants were similar for all compounds.
1
Table 1. J-Values and Chemical Shifts Derived from D
NMR Spectra of Compounds 1−5
compound
Jαβ1, Jαβ2 (Hz)
δ Cγ (ppm)
a
b
Ac-Thr-Pro-NHMe (1)
nd
23.6
c
Ac-Thr-Hyp-NHMe (2)
9.7, 7.8
9.2, 4.6
9.7, 8.1
8.5, 8.5
69.7
d
Ac-Thr-hyp-NHMe (3)
69.5 (67.5)
c
Ac-Thr-[α-(1,4)GlcNAc]Hyp-NHMe (4)
Ac-[α-(1,4)GlcNAc]-Hyp-NHMe (5)
75.7
d
74.8 (74.0)
a
b
Not determined due to overlap between Pro Hα and Thr Hβ. While
We report herein that for a dipeptide that emulates the Thr142
-
two species are evident, the two Cα signals appear to be coincident.
c
On the time scale of the 13C NMR experiment a single, averaged
Pro143 sequence in Skp1 there are noteworthy changes that
correlate with recent findings on the F-box binding capacity of
the isoforms of Skp1.
d
species was observed. The value in parentheses represents a signal
due to the minor conformation.
RESULTS AND DISCUSSION
in a Cγ-exo ring pucker both J-values are in the specific limiting
range of 7−11 Hz, sometimes even converging to produce an
apparent triplet.32,33 In the Cγ-endo ring pucker there are two
quite different dihedral angles between Hα and the neighboring
Hβ protons, giving rise to distinct coupling constants in the
ranges 2−3 and 6−10 Hz. In the case of a twist conformation,
dynamically averaged values of ∼7 Hz are anticipated. Thus, the
pyrrolidine rings in compounds 2, 4, and 5 adopt a Cγ-exo
conformation. The Jαβ values could not be determined for
■
We prepared dipeptides 1−4 (Scheme 2) that track the initial
post-translational modifications of Skp1 and incorporate the
preceding Thr residue. We end-capped the dipeptides to mimic
the amide backbone of the protein and to eliminate
electrostatic effects that arise from ionizable groups.28,29 At
the outset, we did not know the configuration at Cγ of the
hydroxyproline residue and thus included cis-4-hydroxy-L-
proline (hyp) in our repertoire of proline analogues. Specifically
B
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compound 1 due to overlap between Pro Hα and Thr Hβ
signals. For dipeptide 3 containing hyp, the J-values do not fit
either profile but are in agreement with those reported for Ac-
hyp-OMe by Owens et al.34 and may signify a slightly different
conformation, viz. Cβ-exo, Cδ-exo, or related twist confroma-
tions.35
groups N-terminal to the Pro residue have a larger steric bias
toward the trans conformation. This notwithstanding, Kt/c for
dipeptide 1 is low in the context of other dipeptides we have
studied: Ac-Gly-Pro-NHMe (Kt/c 5.5 in D2O at 298 K) and Ac-
Phe-Pro-NHMe (Kt/c 2.2).36 The latter is expected to favor the
cis conformation due to an aromatic−proline interaction.
As anticipated,37,38,41 hydroxylation of the proline residue led
to a significant shift in favor of the trans peptide bond (Scheme
3, Table 2). This arises from stereoelectronic effects that favor a
The electron-withdrawing nature and R-configuration of
substituents at Cγ are attributed with favoring the Cγ-exo
conformation of the pyrrolidine ring and inter alia the trans
conformation of the preceding prolyl peptide bond. The 13C
NMR chemical shift for Cγ has been used to assess the strength
of the electron-withdrawing substituents; our results appear in
Table 1. Hydroxylation at Cγ leads to a significant downfield
shift from 23.6 to 69.7 ppm. Glycosylation further deshields the
Cγ nucleus from the magnetic field, leading to an additional 6
ppm shift downfield upon glycosylation of hydroxyproline, in
accord with the observations of Schweizer and co-workers upon
α-D-galactosylation.27 The J-values and Cγ chemical shifts
reported in Table 1 are very similar to those reported for the
Ac-Pro*-NHMe series,27 implying that the additional Thr
residue does not affect pyrrolidine ring conformation.
The thermodynamics of the cis → trans isomerization were
studied by NMR as described previously.36,37 Kt/c values were
determined at 5° temperature intervals from 25 to 85 °C, by
integration of well-resolved signals for protons associated with
the cis and trans conformations. The derived van’t Hoff plots
are depicted in Figure 2. All peptides demonstrated a decrease
in Kt/c with an increase in temperature, and the linear nature of
the plots indicates that ΔH° and ΔS° are independent of
temperature.
Scheme 3. cis → trans Isomerization of Prolyl Amide Bond
Table 2. Thermodynamics Parameters for Compounds 1, 2,
4, 5, and 7−9
Kt/c
ΔH°
ΔS°
ΔG°(298 K)
compound (298 K) (kcal mol−1
)
(cal mol−1 K−1
)
(kcal mol−1
)
1
2
4
5
7
8
9
2.3
8.7
−0.64
−1.52
−1.73
−0.68
−0.95
−1.33
−1.27
−0.52
−0.80
−0.72
+0.06
−0.87
−1.76
−1.64
−0.49
−1.28
−1.52
−0.70
−0.65
−0.81
−0.78
13.2
3.3
a
a
a
3.2
3.9
3.7
a
Data from ref 27.
Cγ-exo conformation of the pyrrolidine ring that, in-turn, aligns
the peptide backbone for a stabilizing n → π* interaction42,43 in
the trans conformation. Formation of the α-glycoside further
accentuates this effect. This second result contrasts with earlier
reports from Schweizer’s group that showed both α- and β-
galactosylation of Ac-Hyp-NHMe27 or Ac-Hyp-OMe34 to have
essentially no impact on the thermodynamics or kinetics of the
cis → trans equilibrium.
■
◆
▲
Figure 2. Van’t Hoff plots for compounds 1 ( ), 2 ( ), 4 ( ), and 5
( ).
●
To investigate the mechanism behind the differences in
equilibrium constants for compounds 1−5, kinetics of the
prolyl peptide bond isomerization were studied by magnet-
ization transfer NMR experiments.44,45 Experiments were
performed at elevated temperatures (60−80 °C) because at
lower temperatures the rates are too slow to be determined by
this method. Related experiments have been performed in
unbuffered D2O,46−48 and Reimer et al. demonstrated that for
end-capped pentapeptide Ac-Ala-Thr-Pro-Ala-Lys-NH2, the
rate of cis−trans isomerization was independent of pH across
the 1.8−10.8 range and a variety of temperatures.39 This was
shown to be a general phenomenon across a series of
pentapeptides without ionizable side chains. The CIFIT
program49 was used to estimate the rate constants for the
trans → cis process. The reverse reaction rates (cis → trans)
were derived therefrom in conjunction with the equilibrium
constants. Data were fitted according to the Eyring equation50
to afford the activation energy data presented in Table 3.
In the case of the dipeptides, one of the most useful signals in
these studies, vis-a-vis resolution and intensity, was the doublet
̀
at ∼δ1.25 ppm, attributable to the methyl group (Hγ) of the
Thr residue. At lower temperatures, there was excellent
resolution between the upfield (cis) and downfield (trans)
signals. An increase in temperature was accompanied by a move
toward coalescence of the two signals; however, complete
coalescence was not observed within the temperature range
accessible in D2O.38 Broadening and partial coalescence of the
signals in the case of glycosylated dipeptide 4 led to omission of
the 85 °C data point (Figure 2), since integration of the two
signals was not possible.
The incidence of cis prolyl peptide bonds, when preceded by
a Thr residue, has been estimated at 2.9% based on
crystallographic data for proteins.39 In shorter peptides, without
secondary structure, the percentage is higher.40 Larger acyl
C
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a
Table 3. Activation Parameters for trans → cis and cis → trans Isomerization about the Prolyl Amide Bond
trans → cis
cis → trans
c
compound ktc (s−1
)
ΔH‡ (kcal mol−1
)
ΔS‡ (cal mol−1 K−1
)
ΔG‡ (kcal mol−1
)
kct (s−1
)
ΔH‡ (kcal mol−1
)
ΔS‡ (cal mol−1 K−1
)
ΔG‡ (kcal mol−1
)
b
1
2
5
7
8
9
0.38e
0.22e
0.34e
0.31
0.25
0.27
9.86
18.12
18.9
−32.6
−8.3
−5.8
1.4
19.57
20.59
20.63
20.9
0.72f
1.33f
0.94f
0.81
0.73
0.83
9.86
16.96
17.90
20.6
−30.6
−8.0
−6.8
1.2
18.98
19.34
19.93
20.2
b
b
bd
,
21.3
21.1
20.6
bd
,
0.5
21.0
20.2
0.1
20.2
cd
,
1.0
21.3
22.4
6.2
20.6
a
The italic values are data determined in the current work; data below those in italics come from the literature27 for comparative purposes.
Unbuffered D2O. Phosphate buffer, pH 7.4. Reference 27. Measured by magnetization transfer. Calculated from Kt/c and ktc.
b
c
d
e
f
Upon hydroxylation there is a decrease in the rate of
differences in Kt/c, ktc, and kct between compound 5, bearing a
GlcNAc residue, and the Gal derivative 9 reported by Schweizer
and co-workers27 are almost within experimental error. The
only notable difference is in the entropies of activation: the
GlcNAc residue in 5 seems to incur a higher degree of order in
the transition state for peptide bond isomerization than the Gal
residue in 9.
On the basis of GOESY experiments conducted for
compound 9, Owens et al. described a conformation in which
the hydrophobic face of the galactose residue sits over the top
of the pyrrolidine ring27 (Figure 3). There were few crosspeaks
conversion of trans → cis ∼20% in the case of Ac-Pro-NHMe
(8 vs 7)27 and ∼40% at the dipeptide level (2 vs 1). The cis →
trans rates do not follow the same trend; there is a ∼10%
decrease in rate from 7 to 8,27 but a ∼80% increase from 1 to 2.
This accounts for the more pronounced increase in Kt/c in the
dipeptide series. Indeed, Larive and Rabenstein also concluded
that large Kt/c values for prolyl peptide bond isomerization in
oxytocin and arginine−vasopressin were also due to an increase
in the rate of the forward reaction, rather than an inherent
increase in the stability of the trans conformation, relative to
smaller molecules.46 With the increase in electronegativity of
the 4R substituent, there is presumably an increase in the sp3-
character and degree of pyramidalization of the pyrrolidine
nitrogen that facilitates rotation about the C−N amide
bond.47,51,52 As originally demonstrated by Fischer et al.51, a
hydrogen bonding interaction between the pyramidalized
nitrogen and the NH of the residue C-terminal to Pro
(mimicked in our case by the NHMe) helps to lower the
energy of the transition state. Model compounds in which this
amide is substituted for by an ionizable carboxylate or an ester
cannot emulate this feature of the peptide backbone.
In the proline methyl amides 7−9, the free energy barriers to
rotation about the amide bond are dominated by enthalpic
factors. While entropy values are positive and close to zero for
these amino acid derivatives, significant negative entropies of
activation are observed for compounds 1, 2, and 5. This is
generally true of larger peptides46 and reflects a higher degree
of organization in the transition state for an extended peptide
molecule.28 Compound 1 shows very different activation
enthalpy and entropy contributions, relative to compounds 2
and 4. This must be attributable to the influence of the
electron-withdrawing substituent on the energy and degree of
organization of the transition state.
Figure 3. Conformation of compounds 4, 5, and 9. Double-headed
arrows indicate NOE correlations.
in the NOESY spectrum of compound 5, in which Gal is
substituted by GlcNAc. Weak crosspeaks potentially support a
conformation similar to that of 9 but are ambiguous due to
severe peak overlap in the 3.5−4.0 ppm region of the 1H NMR
spectrum. A more defined conformation for dipeptide 4 was
discernible, based on NOESY crosspeaks summarized in Figure
3. One-dimensional GOESY experiments supported these
conclusions (see Supporting Information). As depicted in
Figure 3, it appears that the strong preference for the Cγ-exo
pyrrolidine ring and trans peptide bond (with alignment for the
n → π* backbone interaction) renders the threonine side chain
partially over the pyrrolidine ring, pushing the sugar residue
away.
To better understand the role of the threonine residue, we
considered key chemical shift and coupling constant data for
this residue. In compounds 1, 2, and 4, the Thr Cα and Cβ
chemical shifts did not show much variation: 57.1−57.2 and
67.1−68.8 ppm, respectively. These are somewhat upfield of
statistical average values,56−58 as expected when the (i + 1)
residue is Pro.59 Amide proton (vide infra) and Hα chemical
shifts likewise showed little change upon Cγ substitution: 8.15−
8.26 and 4.58−4.61 ppm, respectively. These values are closer
to those observed for random coil peptides than for α-helix or
β-sheet. motifs.55−57 There was an increase in 3JNH,Hα across the
In the case of glycodipeptide 4 there was no cis/trans pair of
1
resonances in the H NMR spectrum with adequate resolution
for the signal of one conformation to be selectively inverted
without also inverting the signal of the other conformation. Shi
et al. have previously acknowledged that not all peptides are
amenable to rate determination in this manner.47 Lubell and
co-workers have used inversion of the Cα trans signal in 13C
NMR to measure rates of trans → cis isomerization.38,53 In the
present case, a single set of resonances was observed for
compound 4, even at 70 °C. These experimental facts
precluded magnetization transfer experiments that would have
given insight into the impact of glycosylation on cis−trans
isomerization kinetics at the dipeptide level.
The importance of N-acetyl groups of sugars in hydrogen
bonding and peptide conformation has been commented on by
Wong and co-workers16 and also by Live et al.54,55 The
D
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series at 295 K: 4.8 Hz (1), 6.9 Hz (2), and 7.8 Hz (4)
reflecting a change in the theta (ϕ) dihedral angle.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra were acquired in 90% H2O, 10% D2O at
neutral pH. Amide protons were assigned on the basis of
TOCSY spectra. The C-terminal methyl amide (NHCH3) gave
rise to a broad signal with a similar chemical shift in all
compounds. The GlcNHAc amide in compounds 4 and 5
showed a typical 3J coupling contant of 9−10 Hz to H2.60 The
AcNHThr amide (in compounds 1, 2, and 4) displayed a
chemical shift of δ 8.1−8.2 ppm. A significant difference in
chemical shift was observed for the Hδ that is trans to Hγ in
compounds 4 (δ4.17 ppm) and 5 (δ3.79 ppm). This reflects
the relatively deshielded environment in compound 4 where
the polar Thr side chain is in close proximity, compared to
compound 5 where Hδ is shielded by the hydrophobic face of
the sugar. In all cases, variable temperature experiments showed
typical temperature dependence for non-hydrogen bonded
amides (Δδ < −4 ppb K−1). Thus, the strong conformational
preferences appear to be due to steric and stereoelectronic
effects and thereby provide a true baseline for moving forward
to study larger peptides.
Experimental procedures for preparation of compounds 1−6
and associated 1D 1NMR spectra. Integration data for peptides
1, 2, 4, and 5 are tabulated along with calculated
thermodynamics parameters. For compounds 1, 2, and 5,
plots for magnetization transfer experiments are provided along
with calculated kinetics parameters. TOCSY spectra and amide
chemical shift changes as a function of temperature are
provided for compounds 1, 2, 4, and 5. NOESY and pertinent
GOESY spectra are provided for compounds 4 and 5. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
cmtaylor@lsu.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
NIGMS 5R01GM84383-S1.
CONCLUSION
■
Reorganization of the pivotal Pro143 region of Skp1 is a
recurring theme during interactions with F-box proteins.
Recent CD and SAXS studies show that the secondary
structure content of free Skp1 of Dictyostelium is sensitive to
its glycosylation status which correlates with binding to a model
F-box protein.11 Crystal structures have demonstrated a well-
folded state for Skp1 complexed with F-box proteins in other
organisms.19,20 The equivalent of Pro143 is highly conserved
throughout phylogeny except for chordate animals. Even in
organisms with multiple Skp1 genes, at least one copy of the
Pro-containing Skp1 is retained. The amino acid at this position
initiates the C-terminal α-helix. Even in Skp1 proteins where
the equivalent of Pro143 is substituted by another amino acid,
the theme of reorganization persists. A study by Tan et al. of
OCP2 (an orthologue of Skp1 from the organ of Corti)
concluded that binding to OCP1 (bearing an F-box motif) is
accompanied by a substantial degree of refolding.61 The C-
terminal region is alternatively folded in a loop in one
complex.62
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Thus, a picture is emerging that Skp1 exists as an ensemble
of conformations whose suitability to dock with F-box protein
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and deserve further mechanistic studies.
E
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