A b-bulge observed on the edge D b-strand of some b2m crystal structures has been suggested to be crucial in protecting the protein from amyloid aggregation.. Conversely, a straight D-st
Trang 1Stavros Azinas1,2, Matteo Colombo1, Alberto Barbiroli3, Carlo Santambrogio4, Sofia Giorgetti5,6, Sara Raimondi5,6, Francesco Bonomi3, Rita Grandori4, Vittorio Bellotti5,6, Stefano Ricagno1and Martino Bolognesi1
1 Dipartimento di Scienze Biomolecolari e Biotecnologie and CIMAINA, Universita` degli Studi di Milano, Milan, Italy
2 Department of Biochemical Sciences, University of Surrey, Guildford, UK
3 Dipartimento di Scienze Molecolari Agroalimentari, Universita` degli Studi di Milano, Milan, Italy
4 Dipartimento di Biotecnologie e Bioscienze, Universita` di Milano-Bicocca, Milan, Italy
5 Dipartimento di Biochimica, Universita` di Pavia, Pavia, Italy
6 Laboratori di Biotecnologie, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy
Keywords
amyloidosis; 2 microglobulin;
beta-buldge; dialysis related amyloidosis; MHC I;
proline
Correspondence
M Bolognesi, Dipartimento di Scienze
Biomolecolari e Biotecnologie and CIMAINA,
Universita` degli Studi di Milano, Via
Celoria 26, 20133 Milano, Italy
Fax: 00390250314895
Tel: 00390250314893
E-mail: martino.bolognesi@unimi.it
(Received 21 March 2011, revised 29 April
2011, accepted 4 May 2011)
doi:10.1111/j.1742-4658.2011.08157.x
Proteins hosting main b-sheets adopt specific strategies to avoid intermolec-ular interactions leading to aggregation and amyloid deposition Human beta-2 microglobulin (b2m) displays a typical immunoglobulin fold and is known to be amyloidogenic in vivo Upon severe kidney deficiency, b2m accumulates in the bloodstream, triggering, over the years, pathological deposition of large amyloid aggregates in joints and bones A b-bulge observed on the edge D b-strand of some b2m crystal structures has been suggested to be crucial in protecting the protein from amyloid aggregation Conversely, a straight D-strand, observed in different crystal structures of monomeric b2m, could promote amyloid aggregation More recently, the different conformations observed for the b2m D-strand have been inter-preted as the result of intrinsic flexibility, rather than being assigned to a functional protective role against aggregation To shed light on such con-trasting picture, the mutation Asp53fi Pro was engineered in b2m, aiming
to impair the formation of a regular⁄ straight D-strand Such a mutant was characterized structurally and biophysically by CD, X-ray crystallography and MS, in addition to an assessment of its amyloid aggregation trends
in vitro The results reported in the present study highlight the conforma-tional plasticity of the edge D-strand, and show that even perturbing the D-strand structure through a Pro residue has only marginal effects on protecting b2m from amyloid aggregation in vitro
Database Atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession number 3NA4
Structured digital abstract
l Beta-2-microglobulin binds to Beta-2-microglobulin by fluorescence technology (View interaction)
l Beta-2-microglobulin binds to Beta-2-microglobulin by mass spectrometry studies of com-plexes (View Interaction 1 , 2 , 3 )
l Beta-2-microglobulin binds to Beta-2-microglobulin by electron microscopy (View interaction)
Abbreviations
b2m, beta-2 microglobulin; C m , melting concentration; D53P, beta-2 microglobulin Asp53 fi Pro mutant; DRA, dialysis-related amyloidosis; GdHCl, guanidine hydrochloride; MHC-I, class I major histocompatibility complex; TFE, trifluoroethanol; Tm,melting temperature.
Trang 2Intermolecular cross-b interactions are at the basis of
protein amyloid aggregation In the cross-b structure,
b-strands belonging to different protein chains
associ-ate, giving rise to extended intermolecular b-sheets
Because many known protein folds present
solvent-exposed b-strands at the edges of constitutive b-sheets,
the formation of an extended intermolecular b-sheet
structure resulting from cross-b aggregation of protein
chains might turn into a likely event Fortunately, such
a threatening aggregation process is only sporadic
in vivo It has been proposed that proteins adopt
dif-ferent strategies to prevent intermolecular b-b
interac-tions Among these, the b-sheet edge strands tend to
be irregular, generally short or host-charged residues
that make them unsuitable for cross-b interactions,
thus promoting the monomeric state over aggregation
[1] Therefore, introducing a b-bulge in an otherwise
linear edge b-strand may impair the amyloid
aggrega-tion of proteins hosting b-sheets, improving their
solu-bility [1]
Beta-2-microglobulin (b2m) is a 99-residue
b-sand-wich protein that is noncovalently associated as the
light chain of the major histocompatibility complex
class I (MHC I) b2m provides an example of how the
molecular protection strategies described above appear
to be applied; indeed, despite being a fully b-protein, it
is stable as a monomer in solution up to millimolar
concentrations [2,3] In vivo, and under normal
condi-tions, b2m is degraded in the kidneys after dissociation
from MHC-I; however, patients suffering from kidney
failure and undergoing long-term hemodialysis
accu-mulate free b2m in their serum to concentrations that
are ten- to 50-fold higher than physiological
concentra-tions [4] Such stress condiconcentra-tions result in
dialysis-related amyloidosis (DRA), a pathological state
whereby hundreds of grams of b2m amyloid fibrils are
deposited in the skeletal joints and bones, hampering
their functionality [5] Several factors appear to trigger
the formation of b2m amyloid fibrils in vivo, with
par-ticular attention being focussed on the role played by
glycosaminoglycans and fibrillar collagen (type I) [6,7]
Conversely, in vitro, b2m amyloid aggregation can be
achieved using a variety of conditions, such as partial
acid denaturation, proteolytic treatment and the
addi-tion of various chemicals (e.g SDS and
trifluoroetha-nol) [8]
b2m displays a typical immunoglobulin fold, where
two facing b-sheets (composed of strands ABED and
CFG, respectively) are linked by a disulfide bridge
buried in the protein core [9] The D-strand (residues
50–57), which is one of the two edge strands in the
ABED b-sheet, shows a high level of conformational variability in known b2m 3D structures To date, b2m presents a bulged D-strand in all MHC-I crystal struc-tures Asp53 comprises the b-bulge residue at the cen-tre of the D-strand, being involved in two hydrogen bonds to Arg35 of the MHC-I heavy chain [10] Con-versely, in the first reported crystal structure of iso-lated monomeric wild-type b2m, the D-strand is straight, and thus devoid of any b-bulge [11] It was proposed that such a regular edge strand is a structural feature promoting fibril formation [11] Several other crystal structures of isolated monomeric b2m have been determined subsequently and, in every case, the D-strand b-bulge turns out to be absent, regardless of the amyloidogenic propensity tested on each given var-iant [12–16] Conversely, the hexameric structure of the b2m H13F mutant, which is suggested to resemble clo-sely the early amyloidogenic intermediate, presents the Asp53 b-bulge Moreover, NMR studies indicate that the b2m D-strand is very flexible in solution [3,17] Taking all these data together, it was suggested that the variety of conformations observed for the D-strand provide evidence of a high level of structural adapt-ability at this edge strand, and that the straight D-strand conformation trapped in the crystal of mono-meric b2m is not an obligate step in the amyloid for-mation [16]
Despite several recent reports on b2m amyloid fine structure, the location of residue 53 in mature fibrils remains unclear Recent evidence obtained by solid-state NMR suggests that the structural b2m core is conserved in the mature fibrils, whereas some parts of the molecule are reorganized [18,19] However, some-what constrasting evidence on the role of residue 53 is available: Asp53 is predicted to map at the center of a long b-strand [20] and to be involved in intermolecular interactions [19] On the other hand, the side chain of Asp53 was shown to be highly flexible and likely located in a solvent-exposed region [21]
In this context, additional information on the role played by b2m Asp53 bulge during amyloid formation appears mandatory In the present study, we report the structural and biophysical characterization of the b2m D53P mutant, which was engineered with the aim
of determing the role played by the conformational properties of the D-strand in protecting b2m from amyloid aggregation Because Pro residues are known b-breakers, the Aspfi 53Pro mutation was chosen to code for a constitutive perturbation of the D-strand b-structure Our structural and biophysical results show that the D-strand region structure is perturbed
by the mutation, yielding a b2m molecular variant that
is more stable than the wild-type protein We also
Trang 3show that such a region is highly adjustable and that
a shorter and conformationally less regular edge
D-strand does not prevent amyloid fibril formation,
although it does show partly altered aggregation
kinet-ics under specific in vitro conditions
Results
D53P fold stability
To assess the conformational stability of the b2m D53P
mutant, both chemical and thermal protein unfolding
processes were monitored by CD Chemical unfolding at
equilibrium was followed by CD in the far-UV region,
acquiring spectra at increasing guanidine hydrochloride
(GdHCl) concentrations The data indicate that the
D53P mutant is slightly more stable than wild-type b2m
[melting concentration (Cm)D53P= 2.3 m GdHCl
versus Cm wild-type= 2.1 m GdHCl] (Fig 1A); the
cal-culated free energies are: DGðH
2 OÞD53P= 22.9 kJÆmol)1 (m = 9.8 kJÆmol)1Æm)1) and DGðH
2 OÞwild-type= 28.0 kJÆmol)1 (m = 13.2 kJÆmol)1Æm)1) Temperature ramps
monitored by CD in the near- and far-UV regions
con-firm that the mutant is also thermally more stable than
wild-type b2m The secondary structure of the D53P
mutant unfolds at a melting temperature approximately
6C higher than wild-type b2m [melting temperature
(Tm)D53P= 67.4C, Tm wild-type= 61.4C] (Fig 1B)
Similarly, the near-UV spectra revealed a more stable
tertiary structure for the mutant relative to wild-type
b2m (Tm D53P= 65.6C versus Tm wild-type= 62.8C)
(Fig 1C)
Crystal structure of D53P
The structure of the D53P mutant was solved at a
1.9 A˚ resolution, with an Rwork value of 21.4% and an
Rfree value of 26.9% (Table 1); 98 out of 100 amino
acids are traced in the electron density and clear
elec-tron density is visible for Pro53 and the surrounding
region The absence of extensive intermolecular
con-tacts in the crystal lattice confirms that the D53P
mutant was crystallized as a monomeric species, in
agreement with size-exclusion chromatography results
acquired during the mutant purification procedure
(data not shown) The 3D structure of the D53P
mutant matches closely the structure of wild-type b2m,
as observed in the MHC-I (Protein Data Bank code:
2BSS rmsd of 0.74 A˚ for 93⁄ 98 Ca) and displays a
rmsd of 0.85 A˚ for 90⁄ 98 Ca atoms relative to
mono-meric wild-type b2m (Protein Data Bank code: 1LDS)
The latter rmsd value reflects a shift of the AB loop,
which, in the D53P mutant, is tightly packed on the
rest of the protein, as in MHC I (Fig 2A), instead of protruding towards the solvent as in most monomeric b2m structures (e.g the wild-type b2m mutants at resi-due 60) [11,13–16]
Fig 1 (A) Unfolding titration curves (monitored by CD in the
far-UV region) of wild-type (WT) b2m (h) and D53P b2m mutant ( ) as
a function of GdHCl concentration (B, C) Thermal unfolding moni-tored by CD in the far-UV (B) and near-UV (C) regions for wild-type b2m (black) and D53P b2m mutant (gray).
Trang 4The most notable structural adaptations of the
D53P mutant structure are not only found in the
D-strand, as expected, but also in the neighboring
loops By contrast to the previously known b2m
crys-tal structures, where secondary structures are
conserved, the introduction of Pro53 disrupts the
N-terminal part of the D-strand, resulting in a CD
loop that is four residues longer than in wild-type b2m
(residues 43–53⁄ 43–49, D53P ⁄ wild-type, respectively)
(Fig 2); the CD loop is also more exposed than in the
wild-type b2m (Fig 2) The resulting D-strand starts
only after Pro53 but gains one additional residue at its
C-terminus compared to the wild-type protein (residues
54–57⁄ 50–56, D53P ⁄ wild-type, respectively) As shown
in Fig 2B, the DE loop is composed of only three
resi-dues (resiresi-dues 58–60⁄ 57–60, D53P ⁄ wild-type,
respec-tively) and, as a consequence, displays an overall
conformation clearly different from wild-type b2m All
such readjustments affecting the D-strand region result
in the translation of the whole 53–57 segment one
resi-due ‘upstream’, such that resiresi-due ‘n’ of the D53P
mutant matches the site of residue ‘n) 1’ in the
wild-type b2m structure (e.g residue 56 on 55; Fig 2C)
Notably, the region around the mutation site is not
involved in intermolecular contacts to any neighboring
molecule, indicating that the conformation of the
D-strand observed for the b2m D53P mutant is not constrained by crystal contacts
D53P amyloidogenesis in vitro The propensity of the D53P mutant to form amyloid fibrils in vitro was tested with two standard protocols: one at pH 7.4 with 20% trifluoroethanol (TFE) and one at pH 2.5 As shown in Fig 3A,B, the mutant gives rise to amyloid aggregates both at neutral and acidic pH However, at neutral pH, amyloid formation
is delayed by 24 h, although the D53P mutant reaches eventually the same level of aggregation as the wild-type protein; at low pH, the amyloidogenic processes for the two proteins are indistinguishable (Fig 3A)
Oligomerization of natively-folded D53P Similar to a recent report for wild-type b2m and three distinct DE-loop mutants [22], the D53P mutant was analyzed by means of nano-ESI-MS under nondenatu-rating conditions The formation of soluble oligomers was monitored under mild desolvation conditions, aim-ing to favor the detection of noncovalent complexes (Fig 3C) The charge state distribution of the mutant is narrow and consistent with a compact protein confor-mation Spectra deconvolution yields a molecular mass
of 11842 ± 0.5 Da for the D53P mutant, in keeping with the theoretical mass of the engineered protein
As also previously observed, the spectra display con-centration dependence in the explored range (up to
60 lm) [22] As the protein concentration increases, the spectra of the D53P mutant reveal oligomer-specific peaks corresponding to dimers, trimers and tetramers Such concentrations fall below the threshold (100 lm) generally observed for unspecific protein aggregation under electrospray conditions [23]
The nano-ESI-MS data indicate that the Aspfi 53Pro mutation does not prevent the formation
of b2m soluble oligomers Indeed, comparison of the spectra shown in Fig 3C with those of wild-type b2m and the mutated variants previously reported [22] shows that the aggregation propensity of the mutant under nondenaturing conditions is somewhat decreased, although not abolished The effect of the mutation is similar to that observed in the previously tested W60G b2m DE-loop mutant [22] This result is consistent with the structural rearrangements in the
DE loop as a result of the Trp60fi Gly mutation, and with the notion that the DE loop plays an impor-tant role mediating protein–protein interactions [22] These data and other considerations indicate that, under the in vitro conditions tested, the substitution of
Table 1 Data collection and refinement statistics for the crystal
structure of D53P mutant.
b2m D53P mutant
Unit cell edges (A ˚ , degrees) a = 29.1
b = 50.7
c = 71.2
Rmerge(%) 10.6 (56.9)
Refinement
Number of atoms
Ramachandran plot
Values in parentheses are for the highest resolution shell Rmerge=
R |I ) <I>| ⁄ R |I| where I is the observed intensity and <I> is the
average intensity Rwork= Rhkl||Fo| ) |F c || ⁄ R hkl |Fo| for all data, except
5%, which were used for Rfreecalculation.
Trang 5residue 53 with proline and the perturbation of the
b-structure in the D-strand may play only a very
mar-ginal role, if any, in protecting b2m from
oligomeriza-tion of natively-folded molecules
Discussion
The present study reports the design and
characteriza-tion of the D53P b2m mutant, where a Pro residue
was engineered at the center of the D-strand The aim
was to mimic and stabilize the b-bulge at residue 53,
as observed in the b2m crystal structures where the protein is in a nonmonomeric state Because the inser-tion of a proline breaks the regularity of a b-strand as
a result of restrictions of the / and w angles, the resulting (partial) loss of secondary structure was addi-tionally expected to affect the overall b2m stability Nevertheless, the D53P mutant, whose crystal structure indeed shows a shorter D-strand, starting just at Pro53, turned out to be chemically and thermally more stable than wild-type b2m (Fig 1) A possible explana-tion for such unexpected increased stability of the
Fig 2 (A) Left: cartoon representation of wild-type b2m structure in MHC-I complex (Protein Data Bank code: 2BSS) The b-bulge on resi-due 53 breaks the D-strand into two halves Middle: cartoon representation of the D53P mutant with a shorter D-strand and an extended and protruding CD loop Right: monomeric wild-type b2m displays a regular D-strand Residues 53 and 56 are shown as magenta sticks (B) b2m sequence with secondary structures relative to the structure of monomeric wild-type b2m (MON), D53P mutant (D53P), wild-type b2m complexed in MHC I (MHC) and the secondary structures of fibrillar b2m (AMY) predicted by Debelouchina et al [19] b-strands C, D and E are labeled as ssC, ssD, and ssE, respectively The CD and DE loops are marked as lCD and lDE; B, b-bulge Residue 53 is marked as X (C) Stereo representation of strands D and E and the loop inbetween The structures of wild-type b2m in MHC-I (yellow) and the D53P mutant (green) are superimposed.
Trang 6Fibrillogenesis pH 7.4, 20% TFE
Fibrillogenesis pH 2.5
40 50
15 20
20
30
10
0
D53P
0 5
WT D53P
Time (h)
Time (h)
100
5 µ M
15 µ M 90
80 70 60 50 40
Intensity (%) 30 20 10 0
100 90 80 70 60 50 40
m/z
30 20 10 0
1000 1500 2000 2500 3000 3500
100 90 80 70 60 50 40 30 20 10 0
1000 1500 8+
11+
14+17+
2000 2500 3000 3500
1000 1500 2000 2500 3000 3500
100 90 80 70 60 50 40
m/z
30 20 10 0
1000 1500 2000 2500 3000 3500
A
B
C
Fig 3 (A) Fibrillogenesis of D53P mutant Left: kinetics of fibril formation monitored by thioflavin T fluorescence for wild-type (WT) b2m and D53P in 20% TFE at pH 7.4 Right: kinetics of fibril formation at pH 2.5 (B) Transmission electron microscopy images of amyloid fibrils grown in 20% TFE of the D53P mutant (left) and wild-type b2m (right) (C) D53P mutant oligomerization under native conditions monitored
by nano-ESI-MS The most intense peak of the monomer (d), dimer (¤), trimer ( ) and tetramer ( ) is labeled by the corresponding symbol and by the charge state in the final panel.
Trang 7mutant may relate to the high regularity of the D53P
mutant structure (all residues of the refined protein
structure fall in the most favorite regions of the
Rama-chandran plot) Additionally, because Pro residues are
known to decrease the entropy of the protein-unfolded
state, an entropic contribution of Pro53 to the folding
equilibrium, and thus to mutant stability, may also be
considered [24,25]
The amyloid aggregation propensity observed for
the D53P mutant was also rather unexpected The
D53P variant, displays the same kinetics of fibril
for-mation at low pH compared to the wild-type protein,
whereas, in 20% TFE at neutrality, fibril formation is
partly delayed, although, on completion of the
aggre-gation process, the amount of fibrils is similar
(Fig 3A,B) Consistent with these data, under native
conditions and in a concentration-dependent manner,
the D53P mutant spontaneously generates oligomers,
analogously to the wild-type protein (Fig 3C) In
gen-eral, a Pro residue can contribute to inhibit
fibrillogen-esis, as reported for the Alzheimer Ab peptide [26],
islet amyloid polypeptide [27] and mouse
apolipopro-tein A-II [28] By contrast, Pro53 appears to be well
tolerated for b2m fibrillogenesis, which takes place to
the same extent under the in vitro conditions tested but
with somewhat delayed kinetics at 20% TFE (pH 7.4)
Thus, the results for fibrillogenesis in the present study
indicate that a constitutive b-bulge-like structure in the
b2m 50–55 region (a b-sheet edge strand) has a
mar-ginal effect in protecting against aggregation (native or
fibrillar), posing some basic questions on the necessity
of achieving a regular D-strand as a requirement for
the aggregation process
To date, two contrasting hypotheses have been
pro-posed about the regularity of the D-strand in
mono-meric b2m in relation to fibril formation Trinh et al
[11] observed a regular D-strand in the crystal
struc-ture of isolated monomeric b2m, and suggested that
the removal of the Asp53 b-bulge is a necessary
step towards aggregation More recently, Ricagno
et al proposed that occurrence of a regular D-strand
is simply evidence of the flexibility and plasticity of
such a strand, and not directly related to amyloid
for-mation [16] Indeed, recent structural studies have
shown that, regardless of the amyloid propensity of
the mutant considered, several b2m structures display
a straight D-strand, which is regularly
hydrogen-bonded to the neighboring E strand [13–16]
Further-more, two b2m structures showing residue Pro32 in
transconformation, are held to resemble the b2m
amy-loidogenic intermediate [12,29], present different
con-formations of the D-strand: a regular D-strand (in the
P32A mutant) [12] and a bulged D-strand (in the
hexa-meric structure of the H13F mutant) [29] Within the MHC-I complex, the b-bulge centred on b2m Asp53 is favored by hydrogen bonds between Asp53 and resi-due Arg35 of the MHC-I heavy chain [10] Intrigu-ingly, NMR data for monomeric b2m in solution show that the stretch of residues corresponding to the D-strand displays poor b-character, and the D-strand region is highly flexible [3,17] In summary, the b2m D-strand folds as a bulged strand upon interaction with the MHC-I heavy chain, it is flexible in solution and is a regular b-strand when b2m is crystallized as a monomer (Fig 2b) Hence, our D53P mutant crystal structure, together with previously reported structural evidence including the b2m solution NMR structure [3,17], strongly suggests that the D-strand and the neighboring b2m loops are highly flexible⁄ adjustable, and adopt different conformations depending on the structural context
Richardson & Richardson [1] suggested a number of strategies that proteins hosting extended b-structures may adopt to minimize the formation of unspecific intermolecular b-sheets by association of edge b-strands Such strategies can be grouped into two main classes: (a) geometrical irregularities, such as b-bulges, on edge b-strands, hampering intermolecular backbone interac-tions, and (b) inward-pointing charged side chains that become trapped in the hydrophobic core of the fibril upon aggregation Lysines are particularly suitable for such purpose as a result of their charged long and flex-ible side chain; however, other bulky charged residues have been observed in such role Richardson & Rich-ardson [1] show that inward-pointing charged residues
in b-sandwich proteins are often used against amyloid formation In this respect, the locations of Lys91 and His51 within the b2m structure are interesting Lys91
is positioned on the edge G-strand, facing the inner side of the b-sandwich Even though no supporting experimental evidence is available, Lys91 is well posi-tioned to play a protective role against intermolecular cross-b interactions in the native protein On the other hand, the location of His51in the D-strand is intrigu-ing: in native b2m, the His51 side chain points out-wards, although, in the structures of both the P32A mutant and the hexameric H13F mutant, His51 is flipped and points inwards [12,29], a conformation that appears perfectly suited to prevent edge-to-edge aggregation
In conclusion, the crystal structure, fibrillogenesis and biophysical data reported in the present study, when considered in light of the extensive structural literature on b2m, show that the D-strand, at the edge
of the b2m ABED b-sheet, is endowed with wide structural flexibility and plasticity As a result, the
Trang 8different conformations observed in different crystal
structures may simply highlight some of the
conforma-tions that can be accessed by the D-strand and
stabi-lized by a specific structural environment Thus,
straight or bulged D-strands would represent just two
of the possible conformations that are not necessarily
more amyloidogenic or less, respectively, relative to
many others Indeed, we show that the engineering of
a Pro residue at the center of the D-strand (a ‘brute
force’ approach introducing evident D-strand
struc-tural perturbations) has no (protective) effects relative
to the end products of fibrillogenesis, although it
dis-plays some kinetic effects under specific in vitro
condi-tions Indeed, plasticity of the edge D-strand,
facilitating conformational adaptations to the fibril
structural environment, may be one of the leading
fac-tors promoting b2m amyloid aggregation
Because D53P mutant ability to form fibrils is
com-parable to that of the wild-type protein, it is unlikely
that residue 53 in mature fibrils is located within a
long b-strand [19] Rather, we propose that, in mature
b2m amyloid fibrils, residue 53 is located in a loop or
at the end of a strand, where (when mutated to Pro) it
would not interfere with intermolecular association
interactions Such a consideration would be in keeping
with the results reported by Ladner et al [21], who
suggested that residue 53 may fall in a loop region of
the mature amyloid fibrils
Experimental procedures
Mutagenesis
The expression and purification of wild-type and mutant
b2m species was carried out as described previously [30]
Mutagenesis of Asp53 to Pro was performed by using the
QuikChange site-directed mutagenesis kit supplied by
Stratagene (La Jolla, CA, USA) as described previously [13]
The primers used were: forward, 5¢-GAAAAAGTGGAGC
ATTCACCGTTGTCTTTCAGCAAGGAC-3¢; reverse, 5¢-GT
CCTTGCTGAAAGACAACGGTGAATGCTCCACTTTT
TC-3¢
CD spectroscopy
CD experiments were performed on a Jasco J-810
spectro-polarimeter (Jasco Inc., Easton, MD, USA) equipped with
a Peltier device for temperature control Protein was
dis-solved in 50 mm sodium phosphate (pH 7.4) The protein
concentration was 1.4 mgÆmL)1 (1 cm cell path) or
0.1 mgÆmL)1(0.1 cm cell path) for CD measurements in the
near- and far-UV regions, respectively Temperature ramps
were carried out by increasing the temperature from 20C
to 95C at 50 CÆh)1(0.83CÆmin)1) Tmwas calculated as the first-derivatives minimum of the traces recorded in the near- (293 nm) and far-UV (202 nm) regions Chemical unfolding experiments were carried out recording spectra at increasing concentration of GdHCl at 298 K CD signals at
212 nm (i.e the lowest readable wavelength in the presence
of GdHCl) were plotted versus the GdHCl concentration and then fitted with a logistic equation (originlab, version 8.0; OriginLab Corporation, Northampton, MA, USA) The unfolding curves were analyzed using a two-state mechanism Initially, unfolding curves for the NMU transi-tion were normalized to the apparent fractransi-tion of the unfolded form, FU, using the equation:
FU¼ ðY YNÞ=ðYU YNÞ ð1Þ
where Y is the observed variable parameter, and YN and
YU are the corresponding values for the native and fully unfolded conformations, respectively The difference in free energy between the folded and the unfolded state, DG, was calculated by the equation:
DG¼ RT ln K¼ RTln½FU=ð1 FUÞ ð2Þ
where K is the equilibrium constant, R is the gas constant, and T is the absolute temperature The data were analyzed assuming that the free energy of unfolding or refolding, DG, was linearly dependent on the GdHCl concentration [31] (denoted by C):
DG¼DGðH
2 OÞ mC¼mðCm CÞ ð3Þ where DG
ðH 2 OÞ and DG represent the free energy of unfolding or refolding in the absence and presence of GdHCl, respectively; Cm is the midpoint GdHCl con-centration required for unfolding or refolding; and m stands for the slope of the unfolding or refolding curve
at Cm A least-squares curve fitting analysis was used
to calculate the values of DGðH2OÞ, m and Cm
Crystallization and structure determination The D53P b2m mutant was crystalized using the hanging-drop vapor diffusion method The crystallization reservoir was composed of 26% poly(ethylene glycol) 4000, 20% glyc-erol, 0.2 m ammonium acetate and 0.1 m sodium acetate (pH 5.6) The crystal-growth droplet was composed of 1.2 lL of the mother liquor and 1.2 lL of protein solution (8 mgÆmL)1) Crystallization experiments were performed at
294 K Thin needle-crystals grown from a single nucleation site (bushes) were obtained and were flash frozen using mother liquor as cryoprotectant X-ray diffraction data col-lection was performed at 100 K on beamline ID14-2 (ESRF, Grenoble, France) The D53P crystals diffracted up to 1.9 A˚ resolution and diffraction data were processed using mosflm
Trang 9and scala [32,33] Phases were determined by molecular
replacement, using molrep [34] and the b2m W60G mutant
[13] (Protein Data Bank code: 2Z9T) as the search model
The structure was then refined with refmac5 [35] Model
building and analysis was performed with coot [36]
Amyloid fibril formation
Two different protocols were followed for b2m amyloid
aggregation The first protocol was carried out at pH 7.4
by incubation of 100 lm b2m in 50 mm phosphate buffer,
100 mm NaCl (pH 7.4) in the presence of 20% TFE at
37C The second was carried out at pH 2.5 by incubating
100 ll b2m in 50 mm sodium citrate, 100 mm NaCl (pH
2.5) In both cases, 20 lgÆmL)1 of b2m fibril seeds were
added to start the fibrillogenesis Measurements were
con-ducted in triplicate
MS
Nano-ESI-MS was performed on a hybrid
quadrupole-time-of-flight instrument (QSTAR Elite; Applied
Biosys-tems, Foster City, CA, USA) equipped with a nano-ESI
source Metal-coated borosilicate capillaries with a
med-ium-length emitter tip of 1 lm internal diameter (Proxeon,
Odense, Denmark) were used to infuse the samples The
instrumental parameters applied were: declustering potential
80 V; ion spray voltage 1.1–1.2 kV; curtain gas 20 PSI The
interface heater was turned off In the oligomerization
experiments, lyophilized b2m was dissolved in Milli-Q
water (Millipore, Billerica, MA, USA) (at room
tempera-ture) at different final protein concentrations and spectra
were acquired within a few minutes
Transmission electron microscopy
A 10 lL aliquot of suspended b2m amyloids was adsorbed
on 200 mesh formvar⁄ carbon copper grids; after 5 min, the
grids were washed with distilled water to remove buffer
salts and then negatively stained with 2% uranyl acetate
Sample observation was carried over on a EFTEM Leo912
ab (Zeiss, Oberkochen, Germany) transmission electron
microscope at 80 kV, and digital images were recorded by a
Proscan 1K slow scan charge-coupled device (Proscan,
Lagerlechfeld, Germany)
Acknowledgements
This work was supported by Fondazione Cariplo,
Milano, Italy (NOBEL project Transcriptomics and
Proteomics Approaches to Diseases of High
Sociomedi-cal Impact: a Technology Integrated Network), by the
Italian MIUR (FIRB contract RBLA03B3KC_005), by
the EU grant EURAMY and by FAR (Fondo Ateneo per la Ricerca) to R.G We thank Ms Nadia Santo (Cen-tro Interdipartimentale di Microscopia Avanzata, Uni-versity of Milano, Milan, Italy) for technical support
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