However, NMR [8,9] and limited proteolysis [10,11] studies have shown that helix F in apoMb is disordered and readily cleaved by proteases.. The limited proteolysis pattern also led to t
Trang 1effects on the folding pathway within the globin family Luca Codutti1,*, Paola Picotti2,*,, Oriano Marin2, Sylvia Dewilde3, Federico Fogolari1, Alessandra Corazza1, Paolo Viglino1, Luc Moens3, Gennaro Esposito1and Angelo Fontana2
1 Department of Biomedical Sciences and Technologies and MATI Centre of Excellence, University of Udine, Italy
2 CRIBI Biotechnology Centre, University of Padua, Italy
3 Department of Biochemistry, University of Antwerp, Belgium
Introduction
Globins are well-known proteins that share the
charac-teristic of a typical prosthetic group, traditionally
named heme, and corresponding to a protoporphyrin
scaffold carrying a single iron ion, formally in the +2
or +3 oxidation state The metal ion can coordinate several ligands other than protein groups or porphyrin ring atoms Among the exogenous ligands, molecular oxygen has a specific relevance for the function of
Keywords
circular dichroism; globin folding; myoglobin;
neuroglobin; NMR
Correspondence
G Esposito, Dipartimento di Scienze e
Tecnologie Biomediche, University of Udine,
P le Kolbe 4, 33100 Udine, Italy
Fax: +39 0432 494301
Tel: +39 0432 494321
E-mail: rino.esposito@uniud.it
A Fontana, CRIBI Biotechnology Centre,
University of Padua, Viale G Colombo 3,
35121 Padua, Italy
Fax: +39 049 8276159
Tel: +39 049 8276156
E-mail: angelo.fontana@unipd.it
†Present address
Institute of Molecular Systems Biology, ETH
Zurich, Switzerland
*These authors contributed equally to this
work
(Received 15 April 2009, revised 17 June
2009, accepted 15 July 2009)
doi:10.1111/j.1742-4658.2009.07214.x
Neuroglobin is a recently discovered member of the globin family, mainly observed in neurons and retina Despite the low sequence identity (less than 20% over the whole sequence for the human proteins), the general fold of neuroglobin closely resembles that of myoglobin The latter is a paradigmatic protein for folding studies, whereas much less is known about the neuroglobin folding pathway In this work, we show how the structural features of helix F in neuroglobin and myoglobin could represent a pivotal difference in their folding pathways Former studies widely documented that myoglobin lacks helix F in the apo form In this study, limited prote-olysis experiments on aponeuroglobin showed that helix F does not undergo proteolytic cleavage, suggesting that, also in the apo form, this helix maintains a rigid and structured conformation To understand better the structural properties of helices F in the two proteins, we analyzed pep-tides encompassing helix F of neuroglobin and myoglobin in the wild-type and mutant forms NMR and CD experiments revealed a helical conforma-tion for neuroglobin helix F peptide, at both pH 7 and pH 2, absent in the myoglobin peptide In particular, NMR data suggest a secondary structure stabilization effect caused by hydrophobic interactions involving Tyr88, Leu89 and Leu92 Molecular dynamics simulations performed on the apo and holo forms of the two proteins reveal the persistence of helix F in neu-roglobin even in the absence of heme Conversely myoglobin shows a higher mobility of the N-terminus of helix F on heme removal, which leads
to the loss of secondary structure
Abbreviations
Fmoc, 9-fluorenylmethoxycarbonyl; Mb, myoglobin; MbF-P88A, fragment 79–97 of sperm-whale myoglobin with Pro88 replaced by Ala88; MbF-wt, fragment 79–97 of sperm-whale myoglobin; Ngb, neuroglobin; NgbF-A90P, fragment 79–100 of human neuroglobin with Ala90 replaced by Pro90; NgbF-wt, fragment 79–100 of human neuroglobin; NOESY, nuclear Overhauser enhancement spectroscopy; PME, particle mesh Ewald; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy.
Trang 2globins, such as myoglobins (Mbs) and hemoglobins,
which are generally considered to be oxygen storage
and transport proteins [1,2], although other views have
been proposed [3,4] In addition to Mb and
hemoglo-bin, over recent years two additional globins have been
found to occur in a wide variety of vertebrates, namely
neuroglobin (Ngb) [5] and cytoglobin [6] These two
globins exhibit the interesting feature of endogenous
hexacoordination of iron [7], whatever its oxidation
state In brief, the metal ion coordinates the four
pyr-role nitrogens of the porphyrin group and two
imidaz-ole nitrogens of two different histidine residues of the
protein, whereas, in Mbs and nearly all hemoglobins,
only one iron coordination site is given by a histidine
imidazole The two histidines involved in iron
coordi-nation are commonly referred to as proximal and
dis-tal, depending on the relative separation from the
heme metal, and occur at definite locations of the Mb
structural domain The latter consists of eight helices
(A–H) with intervening loops packed with a
character-istic fold of two triple-helix layers with nearly
orthogo-nal relative rotation (three-over-three) (Fig 1) The
heme group accommodates between the two parallel
helices E and F, the proximal histidine being provided
by helix F (residue F8) and the distal histidine being
provided by helix E (residue E7), according to
consen-sus numbering [7] Although the distal histidine
bind-ing to the metal ion is the signature of endogenous
hexacoordination of Ngbs and cytoglobins, only the
proximal histidine coordination occurs invariably in all
globins
ApoMb, the heme-free Mb, retains the highly helical fold of native Mb at neutral pH However, NMR [8,9] and limited proteolysis [10,11] studies have shown that helix F in apoMb is disordered and readily cleaved by proteases The limited proteolysis pattern also led to the establishment that no other potential cleavage sites
of apoMb undergo hydrolysis, as a consequence of the stability of the helical fold of the protein [11] An important determinant responsible for the conforma-tional flexibility of the chain segment encompassing helix F in apoMb has been recognized in the nature of residue F3, a proline residue that disrupts the local a-helical conformation and destabilizes significantly the whole helix F (Fig 1) Indeed, substitution of the helix-breaking Pro88 (F3) residue with the helix-forming ala-nine residue in sperm-whale apoMb successfully meets expectations and apparently restores the local helix geometry, as inferred from CD profiles and limited proteolysis [11], although none of the techniques can distinguish partial from full restoration In these earlier studies [8,11], it was proposed that helix F in the native holoprotein is stabilized by interactions with the heme moiety, counterbalancing the helix-breaking effect of proline As a proline residue at location F3 occurs in more than 90% of Mb sequences and several hemoglo-bin chains, these glohemoglo-bin species in their apo form should exhibit low, if any, helical propensity in the corresponding helix F segment This is in agreement with the proposed main folding pathway of apoMb at neutral pH, sketched as U fi AGH fi ABGH fi ABCDEGH fi N, where U and N are the unfolded
C
Fig 1 Three-dimensional structure of human Ngb (A) and sperm-whale Mb (B) The models were constructed from the X-ray structure of the Ngb mutant C46G ⁄ C55S ⁄ C120S (PDB code 1OJ6, chain B) and sperm-whale Mb (PDB code 1VXD) Helix F is highlighted by a white ellipsoid in both diagrams (C) Sequences of the pep-tides that were addressed in the present study, i.e helix F encompassing fragments 79–100 of Ngb and 79–97 of sperm-whale
Mb, together with the corresponding vari-ants A box highlights the actual extension
of helix F in the parent protein structures The wild-type sequences are indicated as NgbF-wt and MbF-wt; the variant sequences are identified by the correspond-ing mutations, i.e NgbF-A90P and MbF-P88A, respectively.
Trang 3and native conformations, respectively, and A, B, etc.
are the helix segments [12] The folding scheme confirms
the absence of a stably folded helix F in native apoMb,
in contrast with the N state of the holoprotein [12]
The stability of the globin domain has been
addressed recently by comparing the apo forms of
horse Mb and human Ngb at acidic pH (P Picotti,
unpublished results) The well-established instability of
apoMb at low pH [13,14] was confirmed At variance
with the extensive loss of apoMb secondary structure,
the apoNgb chain was observed to preserve most of
the helical fold at acidic pH and limited proteolysis
experiments suggested that only the N-terminal
frag-ments were sufficiently flexible to become susceptible
to proteolytic cleavage Therefore, among the
pre-served helical segments of apoNgb, there was also
helix F This finding appears to be consistent with the
absence of a proline residue in the chain segment
encompassing helix F in apoNgb (Fig 1)
A useful approach to the study of the mechanism
of protein folding entails the analysis of the
confor-mational preferences of isolated peptide fragments
Short linear peptide fragments cannot exhibit the
tertiary interactions that they establish in the intact
proteins Therefore, the assessed conformational
trends of isolated fragments are the same as those
occurring in the protein chain during the early stages
of folding, when only local and inherent
conforma-tional propensities drive the folding process Along
these lines, a direct CD and NMR investigation was
carried out previously in order to establish the folding
propensities of Mb peptide fragments [15] In addition
to confirming inherent helix propensities for AB and
GH segments, this study also showed that the helix F
fragment has quite a low propensity towards helical
geometry, even in the presence of
2,2,2-trifluoro-ethanol (TFE)
In order to investigate more deeply the suggested
differences in helix F stability within the globin family,
limited proteolysis of apoNgb at neutral pH and the
intrinsic conformational stability of peptides
encom-passing the different helix F variants (Fig 1) are
addressed here Indeed, it is shown that helix F in
apo-Ngb has a strong propensity for a-helical secondary
structure, at variance from helix F in apoMb These
results allowed us to infer that the folding pathway of
apoNgb is different from that of apoMb, despite the
similarity of their overall fold The conclusion reached
in this study reinforces the view that the same protein
structural topology does not imply the same folding
pathway An analogous view was also expressed in a
comparative study of the folding pathway of Mb [12]
and leghemoglobin [16]
Results and discussion Limited proteolysis
Limited proteolysis experiments were performed on wild-type human apoNgb at neutral pH with the enzyme thermolysin (Fig 2) Compared with the incu-bation times typically required by horse or sperm-whale apoMb, i.e seconds [10,11], apoNgb proteolysis proved to be much slower After 4 min of incubation, apoNgb shows only two sites of preferential cleavage, i.e at the level of the N-terminal helix (helix A), pre-cisely between Ala15 and Val16, and at the interhelical segment between helix F and helix G, precisely between Ala98 and Val99 The proteolysis pattern of the latter region also involves hydrolysis between Ser91 and Leu92, with the formation of fragment 16–91, which becomes the predominant species at longer protease incubation times The later onset of fragment 16–91 demonstrates that it derives from further proteo-lytic digestion of the initially formed species 16–98 at the level of the newly exposed C-terminus However, a
Fig 2 Limited proteolysis of human apoNgb at neutral pH Proteol-ysis of apoNgb by thermolysin (enzyme to substrate ratio 1 : 100
by weight) was conducted at 25 C in 50 m M Tris-HCl, 0.15 M NaCl,
pH 7.0 The proteolysis mixture was analyzed by reverse-phase HPLC after 4 and 30 min of incubation The identities of the protein fragments were established by electrospray mass ionization mass spectrometry and are indicated by the labels near the chromato-graphic peaks.
Trang 4significant amount of undigested full-length protein is
still present at either proteolysis intervals Therefore,
the cleavage location is partially dependent on the
incubation time of the substrate with the protease, at
least within the limits of the experimental protocol
Cleavage at the level of the turn-like fragment, joining
helix F to the rest of the C-terminal region, suggests
that this region is highly flexible in Ngb at neutral pH,
and thus it is a proper protease substrate The
addi-tional cleavage site at the N-terminal region of Ngb
(at the end of helix A) recalls the previously observed
limited proteolysis pattern of apoMb at acidic pH,
with a cleavage extended to most of helix B at low pH
[13] Strikingly, at both pH conditions, helix F of
human apoNgb does not undergo proteolytic cleavage
as instead observed in apoMbs, under either neutral
[10,11] or acidic conditions [13], suggesting that the
apoNgb helix F maintains a sufficiently rigid structure
to prevent proteolysis These results are perfectly in
line with the agadir [17] secondary structure
predic-tions reported in Fig 3, which illustrates the helical
propensity for the whole Ngb sequence, at neutral
and acidic pH, and for wild-type or mutated helix F
peptides of Ngb and Mb
CD analysis
Far-UV CD measurements (Fig 4) of the peptides
under investigation were conducted in different
experi-mental conditions in order to analyze their content of
secondary structure The investigated peptides
encom-pass the native sequences of helix F in sperm-whale
Mb (MbF-wt) and human Ngb (NgbF-wt) and the
corresponding mutants obtained by the replacement of
Pro88 (ProF3, in the globin consensus map [7]) with
an alanine in Mb (MbF-P88A), and of Ala90 (AlaF2)
with a proline in Ngb (NgbF-A90P) (see Fig 1) Fig-ures 4A,B depict the spectra obtained for NgbF-wt at neutral pH conditions with increasing amounts of TFE and in aqueous solution at decreasing pH, respectively
At neutral pH, the CD spectrum of NgbF-wt displays two prominent minima at 208 and 222 nm, typical of a-helical polypeptides The helix content of NgbF-wt steadily increases with TFE, from 35% without organic solvent to 56% at 20% TFE On lowering the
pH (Fig 4B), instead, the helix content decreases from the same initial value as in Fig 4A to 30% at pH 2.2 The mutation of Ala90 into proline destroys the helix content of the parent sequence, as evident from the corresponding CD spectrum typical of random coil peptides (Fig 4C) The addition of TFE restores some helix content (17%) in NgbF-A90P, to an extent, how-ever, much below that observed for NgbF-wt Far-UV
CD spectra collected on the peptides encompassing the sequence of helix F in Mb are reported in Fig 4D As expected from previous results on the whole protein and mutants thereof [11], as well as on isolated frag-ments [15], the peptide MbF-wt, with the natural sequence bearing a proline in position F3, displays very little helical content at neutral pH, whereas the peptide MbF-P88A, where the proline is replaced by alanine, exhibits a slightly higher helix content (16%)
It is worth noting that the experimental helix content obtained from CD data for the isolated peptides paral-lels the expectations obtained using agadir semi-empirical predictions on the corresponding native and mutant full-length proteins (Fig 3, right)
NMR analysis
1H NMR spectra were collected only for the NgbF-wt fragment at two different pH values, i.e pH 6.3 and
Ngb, pH 2.0
Ngb, pH 7.0
Mb P88A
Ngb WT
Fig 3 Left: helical propensity of the poly-peptide sequences of Ngb at neutral and acidic pH calculated using the AGADIR algo-rithm [17] The locations of the eight helices (A–H) along the polypeptide chain of the protein are also indicated by boxes, accord-ing to the structural features obtained from the PDB record Right: AGADIR -predicted heli-cal propensities for helix F of wild-type human Ngb, A90P human Ngb, wild-type sperm-whale Mb and P88A sperm-whale Mb.
Trang 5pH 2.1 in water, and pH 6.3 in 10% aqueous TFE.
Detailed analysis was performed, however, only for the
datasets obtained in water to which we will refer,
unless otherwise indicated Spin systems were first
identified, for both pH conditions, in the total
correla-tion spectra, and then assigned on the basis of nuclear
Overhauser enhancement spectroscopy (NOESY) map
sequential connectivity patterns [18] A general
over-view of the NMR information shows that, for both
series of experiments, no long-range restraints
(interac-tions between nuclei more than five residues apart)
were detected Figure 5 displays the distribution along
the sequence of all collected restraints under both
experimental conditions Secondary structure
meaning-ful cross-peaks have been found for both pH
condi-tions and are shown in Fig 6 Even a cursory
examination suggests that typical helix conformational
patterns occur in the central region of the investigated
peptide at both pH conditions, whereas the N-terminal
and C-terminal segments appear to be poorly
struc-tured An additional interesting feature that emerges from nuclear Overhauser enhancement restraints is the occurrence, at both pH conditions, of medium-range hydrophobic interactions between Tyr88 Hd or Heand Leu85, Leu89, Leu92 Hd
After removing all redundancies, the experimental restraint sets consisted of 211 meaningful interatomic distances for experiments made at pH 6.3, and 236 meaningful interatomic distances for experiments made
at pH 2.1 The two series formed the experimental databases for the subsequent restrained modeling Table 1 summarizes the final output of restrained modeling
Structural validation performed using the software aqua and procheck-nmr [19] confirmed the presence
of a regular a-helix secondary structure at both pH conditions, with slightly different lengths At pH 6.3, the a-helix involves residues from Glu86 to Leu92, whereas, at pH 2.1, the a-helix extends from Glu87 to Ser91, in qualitative agreement with the estimates
10
NgbF, pH 7.2
–10
0
0% TFE
10% TFE
–20
20% TFE
–3 (deg·cm
2 ·dmol
Wavelength (nm)
200 210 220 230 240 250
0
NgbF
–10
–5
pH 2.2
pH 7.2
pH 4.1
–15
–3 (deg·cm
2 ·dmol
Wavelength (nm)
200 210 220 230 240 250
NgbF, pH 7.2
–5 0
–15
–10
NgbF-A90P NgbF-A90P, 20% TFE
–3 (deg·cm
2 ·dmol
Wavelength (nm)
200 210 220 230 240 250
MbF, pH 7.2
MbF-P88A
–3 (deg·cm
2 ·dmol
–5 0
–20 –15
–10
MbF
Wavelength (nm)
200 210 220 230 240 250
Fig 4 CD characterization of peptides
encompassing helix F of human Ngb and
sperm-whale Mb (see Fig 1) (A) Far-UV CD
spectra of NgbF-wt peptide dissolved in
50 m M Tris-HCl ⁄ 0.15 M NaCl, pH 7.0, in the
presence of different amounts of TFE (B)
Far-UV CD spectra of NgbF-wt peptide
dis-solved in 10 m M HCl, pH 2.2 or pH 4.1 The
spectrum at pH 7.2 is redrawn for
compari-son (C) Far-UV CD spectra of NgbF-A90P
peptide dissolved in 50 m M Tris-HCl ⁄ 0.15 M
NaCl, pH 7.0, in the presence of 20% TFE.
(D) Far-UV CD spectra of MbF-wt and
MbF-P88A peptides dissolved in 50 m M
Tris-HCl ⁄ 0.15 M NaCl, pH 7.0 All spectra
were recorded at 25 C.
Trang 6obtained from CD data Over these fragments, the
average upfield deviations of Ha chemical shifts from
the values of statistically disordered structures [20] are
0.21 ± 0.11 and 0.19 ± 0.13 p.p.m under neutral and
acidic conditions, respectively (the corresponding value
in TFE is 0.25 ± 0.12 p.p.m.) Such deviation extents
are above the chemical shift index threshold to validate
helical tracts [21], which, for a linear peptide in water,
suggests that a helical geometry is locally significantly
populated Validation of the secondary structure for
the remaining residues in both pH conditions
con-firmed a statistically disordered state According to
procheck-nmr, at both pH conditions, refined
struc-tures showed no dihedral angle of the fragment 86–92
in disallowed Ramachandran regions The helical
seg-ments also revealed low accessibility because of a
Residue number
Residue number
A
B
Fig 5 Restraint distribution along the sequence of the NgbF-wt
peptide The restraints obtained at pH 6.3 (A) and pH 2.1 (B) and
subsequently used for simulated annealing calculations are given.
In the histograms, white represents intraresidue restraints, light
grey sequential restraints and dark grey medium-range restraints.
Fig 6 Secondary structure diagnostic restraints obtained at pH 6.3 (A) and pH 2.1 (B) The bar thickness is proportional to the corre-sponding nuclear Overhauser enhancement intensity.
Table 1 CYANA 2.1 and DISCOVER output parameters for NgbF-wt restrained molecular dynamics calculations and subsequent refinement.
CYANA
Average backbone rmsd to mean ⁄ 10)1nm
Average heavy atom rmsd
to mean ⁄ 10)1nm
Target function ⁄ 10)2nm 2 (6.23 · 10)2) ±
(1.85 · 10)2) Violated distance
constraints
0 Violated van der Waals’
constraints
0
DISCOVER
Average backbone rmsd
to mean ⁄ 10)1nm
Average heavy atom rmsd
to mean ⁄ 10)1nm
CYANA
Average backbone rmsd
to mean ⁄ 10)1nm
Average heavy atom rmsd
to mean ⁄ 10)1nm
Target function ⁄ 10)2nm 2 (0.28 ± 4.53) · 10)2 Violated distance
constraints
0 Violated van der Waals’
constraints
0
DISCOVER
Average backbone rmsd to mean ⁄ 10)1nm
Average heavy atom rmsd
to mean ⁄ 10)1nm
Trang 7back-fold trend of the disordered flanking regions
(Fig 7) Additional validation parameters are reported
in Table S1 (see Supporting information)
A fitting of the 20 final conformers (at both pH
con-ditions) over the a-helix validated zone led to mean
backbone rmsd deviation values of 0.037 ± 0.021 nm
for structures calculated at pH 6.3 and
0.026 ± 0.013 nm for structures calculated at pH 2.1
Additional details are given in Table 1
Figure 8 shows a diagrammatic representation of the
superposition of the structure of family conformers at
both pH conditions The structures were superimposed
to minimize rmsd within the regular helical fragments
in both cases As apparent from the side-chain
distri-bution, it is likely that hydrophobic interactions
between Tyr88 and both Leu89 and Leu92 side-chains
create a scaffold capable of stabilizing the local helical
fold in either pH conditions Indeed, the chemical
shifts of the Leu92 side-chain isopropyl moiety are
shifted upfield by 0.13–0.18 p.p.m., whereas Leu89 Hb
resonances occur downfield with respect to the basic
aqueous shift value [20] by 0.18–0.20 p.p.m at both
pH conditions
Molecular dynamics simulations Snapshots have been taken at 100 ps intervals in order
to obtain a statistical ensemble for the three systems studied We consider first the molecular dynamics sim-ulations of the apo forms of Mb and Ngb in order to check whether any difference in dynamics could be highlighted even in a simulation time as short as 3 ns Although the loss of secondary structure for apoMb
is expected, it is not obvious how fast this process may
be Molecular dynamics simulation shows that the N-terminal part of helix F (entailing residues Glu83 to Leu86) loses its helical conformation in the first 200 ps
of simulation In particular, the u and w angles formed
by these residues are quite different from those of stan-dard a-helices and exhibit very large fluctuations There is no clear conformational transition towards completely different conformations, but overall the backbone is very flexible The results concerning helix
F are in agreement with earlier simulation studies [22,23] This picture is further confirmed by the
analy-A
B
Fig 7 Overlay of the 20-membered conformer families of the
NgbF-wt peptide Superpositions were obtained by fitting the
struc-tured regions observed at different pH conditions: (A) pH 6.3; (B)
pH 2.1.
A
B
Fig 8 A diagrammatic view of the structured regions of the 20 peptide family members at both pH conditions: (A) pH 6.3; (B) pH 2.1 The side-chains involved in the a-helical secondary structure are highlighted in red.
Trang 8sis of backbone rmsds, which are larger than 0.2 nm at
residues 83 and 84
In contrast, apoNgb maintains a standard a-helical
conformation at the corresponding residues (Glu86 to
Leu89) The a-helix does, however, start for most of
the time at residue Ser83 The hypothesis put forward
in this work is that Tyr88 plays a crucial role in
scaffolding residues 85, 89 and 92 Indeed, when these
residues are superimposed, the average heavy atom
rmsd between any two pairs of snapshots is 0.088 ±
0.026 nm, therefore showing a rather stable
arrange-ment of these amino acids This rmsd value can be
compared with that obtained for the corresponding
residues in apoMb, which is 0.126 ± 0.042 nm In
order to check that the loss of ordered conformation in
apoMb was not a simulation artifact, a 3 ns molecular
dynamics simulation was performed on the holo form
of the same protein In this simulation, the helical
con-formation is preserved, as expected during all
simula-tions, because of the additional constraint on the helix
provided by the covalently bonded heme group
In order to further validate the NMR results, 1.2 ns
molecular dynamics simulations were run on the
stud-ied peptides The results further confirmed all the
available evidence For the MbF-wt fragment, the
heli-cal conformation is lost after 500 ps at the N-terminal
residues and the helix is found mostly between residues
Leu86 and Ala94 The missing backbone amide proton
of proline seems to be the determinant of secondary
structure loss at the N-terminus, according to the
typical CO(i)–HN(i + 3), CO(i)–HN(i + 4)
hydrogen-bonding pattern of helical conformations In contrast,
for the NgbF-wt peptide, the regular helical
conforma-tion is maintained after 500 ps, from Ser83 to Val99
Interactions among hydrophobic moieties of Leu85,
Leu89, Tyr88 and Leu92 appear to be particularly
rele-vant in conferring stability to the helix
Ngb helix F
The collection and interpretation of structural data at
neutral and acidic pH conditions highlight the
interest-ing features of human Ngb helix F structurinterest-ing
Previ-ous evidence has shown that helix F of apoNgb is
preserved from proteolysis at pH 2 (P Picotti,
unpub-lished results), suggesting the conservation of its
sec-ondary structure in spite of the extreme conditions By
contrast, at the same pH value, apoMb underwent
extensive proteolysis [13], whereas, at neutral pH,
proteolytic cleavage occurred only at helix F [10,11] In
the present work, a clear persistence of the a-helical
structure in strong acidic conditions has also been
con-firmed for the isolated NgbF-wt peptide A first glance
at the amino acid charge position over this peptide sequence led us to postulate initially a secondary struc-ture stabilization as a result of favorable charge inter-action with the a-helix macrodipole [24,25] of Glu86 and Glu87 side-chains Hence, decreasing the pH to a value of 2.1 should have affected the whole helix stability because of a loss of the side-chain-mediated electric shielding from Glu86 and Glu87 carboxylates
As mentioned previously, a decrease in pH decreases the a-helix extension, from residues 86–92 to 87–91, but does not totally disrupt it This means that, in the addressed sequence, the main helix-nucleating driving forces are likely to arise from other structuring energy contributions One such contribution may arise from the interactions that could be established in NgbF-wt between Glu86 and Glu87 amides and the carbonyl and side-chain oxydryl acceptors of the preceding serine pair, in particular Ser84 occurring in the ideal position for N-capping [26] (no similar N-capping potentiality is present in MbF-wt) However, our exper-imental evidence does not support this N-capping occurrence in NgbF-wt, but rather hydrophobic inter-actions Medium-range interactions revealed by NMR NOESY spectra at both pH conditions involve princi-pally Tyr88, Leu89 and Leu92, arranged in a helical geometry with an ideally suited separation between Tyr88 and Leu92 This experimental evidence is compatible with the hydrophobic scaffold-mediated hypothesis advanced above
The limited proteolysis pattern observed for apoNgb after 30 min of incubation with thermolysin is in line with this interpretation Indeed, the proteolytic cleav-age affecting helix F, which is unprotected as a result
of the loss of segment 99–151, occurs between residues Ser91 and Leu92, i.e at the C-end of the proposed hydrophobic scaffold, despite the fact that an even more favorable thermolysin proteolytic site can be rec-ognized between Tyr88 and Leu89 Interestingly, equivalent results were also observed when thermolysin hydrolysis was performed on the isolated NgbF-wt peptide (Fig S3, see Supporting information) Indeed,
by aligning the known Ngb sequences obtained from the UniProtKB⁄ Swiss-Prot database (http://www expasy.org/cgi-bin/get-similar?name=globin%20family),
a general motif can be recognized to occur in all F-helices: {L82 -[SH]-[ST]-L-E-[ED]-[YF]-L-X-X-L-G-[R,K]-K-H-[R,Q]-A98} In addition to the invariant His96 (proximal HisF8), which is expected because of its essential role in heme coordination, Leu92 (LeuF4)
is also well conserved in the globin family [27] and, indeed, it has structural relevance in maintaining the position of the His96 imidazole ring [7] In addition, the Ngb subfamily is specifically characterized by the
Trang 9occurrence of a conserved aromatic residue at position
88 (tyrosine or phenylalanine) The recognized motif
also presents phylogenetic persistence of negatively and
positively charged (at physiological pH) clusters close
to the N- and C-termini of the helix, respectively
Finally, the presence of conserved leucines, three to
four residues apart, is most noticeable, an arrangement
that creates, with the mentioned aromatic residue at
position 88, the hydrophobic face of an amphipathic
helix (see Fig 9) The regularly spaced leucine residues
are likely to contribute to the extension of the helix F
N-terminal side via hydrophobic stacking This is
con-sistent with the NMR evidence obtained for NgbF-wt
in aqueous TFE (10%), which suggests some
propen-sity to helix elongation, namely to a helix also
involv-ing the N-terminal fragment 79–85 (Figs S1 and S2,
see Supporting information), in agreement with the
conspicuous helix content increase also observed by
CD under similar conditions Inspection of the crystal
structure of human Ngb reveals the relevance of the
helix F amphipathicity Although the heme surface
contacts the upper side of the helix F hydrophobic
face, a crucial contact involves Leu89, i.e the first
leu-cine of the helix F hydrophobic scaffold, and Met144
of helix H (Cc89–Cc144= 0.441 nm) As Met144 is
invariant within Ngb sequences, it can be proposed
that the positioning of the helix F hydrophobic
scaf-fold may be dictated by helix H, i.e a strongly
persis-tent secondary structure element that has always been
recognized to be involved in the early folding events,
at least in Mb [12] and leghemoglobin [16]
Conclusions The inherent conformational properties of isolated protein fragments have often been used to analyse pro-tein folding pathways [28] As fragments cannot develop the long-range interactions of native proteins that usually form along the folding pathway of the whole protein chain, the propensity of a protein frag-ment to adopt a precise secondary structure appears to
be relevant to the early protein folding events The results of this study indicate that a peptide encompass-ing helix F of Ngb has a strong propensity to adopt
an a-helical secondary structure in water solution, as given by far-UV CD and NMR measurements As the isolated Ngb helix F autonomously forms a hydropho-bic helical scaffold, we advance the hypothesis that hydrophobic interactions within the core segment 88–92 of Ngb helix F could represent the primary helix-forming driving force that contributes to the ini-tial helix core during the folding of apoNgb In partic-ular, the presence of a conserved tyrosine residue at position 88 appears to provide a very stable arrange-ment of nearby residue side-chains in helix F, thus making the helical geometry quite stable In addition, the hydrophobic scaffold 88–92 appears to be suitably located to establish a favourable hydrophobic contact with a conserved residue of helix H, probably an early-folding one by analogy with previous evidence [12,16] Although helix F formation seems to occur early in the folding pathway of Ngb, all models of apoMb folding pathways so far developed do not include the structuring of helix F [16,23,29–31] Molecular dynam-ics simulations also support this difference Therefore,
we suggest a different folding pathway for Ngb and
Mb, with helix F being an early nucleating folding core in Ngb, rather than the last folding step as in
Mb, where helix F is formed only on addition of the heme moiety
The globin family has been used previously as an excellent experimental system for analysing protein folding mechanisms, as the helical globin fold is highly conserved between proteins with widely differing amino acid sequences [32] It has been proposed that the folding pathways of evolutionarily related proteins with similar three-dimensional structure, but different sequences, should be similar [33,34] Mb and Ngb share an almost superimposable three-dimensional fold and show a low degree of sequence identity (less than 20% over the whole sequence for the human proteins)
In this study, we conclude instead that, despite the
Fig 9 A ball-and-stick view of the crystallographic structure of
human Ngb helix F with a heme ring Hydrogen bonds are
high-lighted in green with the corresponding distances calculated by
Swiss PDB viewer.
Trang 10strong similarity of the overall fold of Mb and Ngb,
these two proteins display different folding pathways
A similar scenario has emerged already from the
com-parison of Mb and leghemoglobin, two proteins with
the same type of folded structure, but adopting
differ-ent folding pathways [16] These two proteins form
rapidly compact helical folding intermediates that
direct the overall folding pathway of the whole
poly-peptide chain, but the details of the pathways are
dif-ferent and depend on the local amino acid sequences
Although apoMb forms an A(B)GH helical
intermedi-ate [29], leghemoglobin initially forms an intermediintermedi-ate
given by helices G and H and part of helix E [16]
Moreover, recently, it has been shown that the
molecu-lar details of the intermediate formed by
leghemoglo-bin in kinetic experiments differ from those of the
equilibrium molten globule intermediate [35]
There-fore, individual proteins, such as Ngb, Mb or
leghemo-globin, despite their overall fold similarity, can follow
different folding pathways dictated by the solution
conditions and differences in amino acid sequences
[36]
Experimental procedures
Materials
Thermolysin from Bacillus thermoproteolyticus was
pur-chased from Sigma (St Louis, MO, USA) Solvents, resin
and coupling reagents for peptide synthesis were obtained
from Applied Biosystems (Foster City, CA, USA) All
pro-tected amino acids were purchased from Novabiochem
(Laufelfingen, Switzerland) HPLC-grade solvents were
obtained from Merck (Darmstadt, Germany)
The expression and purification of the Ngb mutant
C120S was performed as described previously [37] The
Cys120 to serine replacement in Ngb was made in order to
avoid protein aggregation processes of the apo form of the
protein (apoNgb) as a result of the formation of an
inter-molecular disulfide bond The preparation of apoNgb was
obtained from the corresponding holoprotein by the
removal of heme by reverse-phase HPLC separation
Briefly, the holoprotein was loaded onto a C18 Vydac
col-umn (4.6· 250 mm; The Separations Group, Oak Ridge,
TN, USA), eluted with a linear gradient of
water–acetoni-trile, both containing 0.05% (v⁄ v) trifluoroacetic acid
(TFA), from 5 to 40% in 5 min and from 40 to 60% in
25 min, at a flow rate of 0.8 mLÆmin)1 The effluent was
monitored by absorption measurements at 226 nm and
fractions containing the protein were pooled and then
con-centrated in a SpeedVac system The possible
contamina-tion of the apoprotein preparacontamina-tion by the holoprotein was
assessed spectrophotometrically, and no significant
absorp-tion was observed in the Soret region
Peptide synthesis The peptides used in this study were designed to reproduce chain segments 79–100 of human Ngb and 79–97 of sperm-whale Mb and were produced as N-acetylated and C-ami-dated species In addition to the wild-type peptides, two variants were also studied bearing a single residue replace-ment The amino acid sequences of the peptides used herein are shown in Fig 1B The peptides were synthesized by solid-phase peptide synthesis using an automated peptide synthesizer (model 431-A; Applied Biosystems) The 9-flu-orenylmethoxycarbonyl (Fmoc) strategy was used through-out the peptide chain assembly [38] As solid support the 4-(2¢,4¢-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyace-tamido-norleucylaminomethyl resin (Rink amide AM resin) (Novabiochem) (loading of 0.74 mmmolÆg)1) was used The side-chain-protected amino acids used were as follows: Fmoc-Asp(tert-butyl), Fmoc-Glu(tert-butyl), Fmoc-Ser (tert-butyl), Fmoc-Thr(tert-butyl), Fmoc-Tyr(tert-butyl), Fmoc-Gln(trityl), Fmoc-His(trityl), Fmoc-Lys(tert-butyloxy-carbonyl) and Fmoc-Arg(2,2,4,6,7-pentamethyldihydroben zofuran-5-sulfonyl) Coupling was performed with a single reaction for 45 min by a 0.45 m solution in N,N¢-dimethyl-formamide of 2-(1-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-nium hexafluorophosphate and N-hydroxybenzotriazole in the presence of N-ethyldiisopropylamine, following the man-ufacturer’s protocols At the end of the solid-phase synthe-sis, the peptidyl-resins were acetylated by treatment with 10% acetic anhydride in N,N¢-dimethylformamide to yield
an N-acetylated peptide Cleavage of the crude peptides was performed by reacting the acetylated peptidyl-resins with TFA–H2O–thioanisole–ethanedithiol–phenol (10 mL : 0.5 mL : 0.5 mL : 0.250 mL : 750 mg) for 2.5 h The peptides were pre-cipitated with ice-cold ethyl ether and isolated by centrifuga-tion The pellets were washed several times with ether, dissolved in water and lyophilized Crude peptides were purified by a preparative reverse-phase HPLC column (PrepNova-Pak HR C18, 250 mm· 10 cm, 6 lm bead size; Waters, Milford, MA, USA) at 12 mLÆmin)1using a linear gradient of 5–50% acetonitrile in 0.08% TFA The molecu-lar masses of the peptides were confirmed by electrospray mass ionization mass spectrometry using a Micro Q-Tof mass spectrometer (Waters, Manchester, UK) The purities
of the purified peptides were 98% as evaluated by analytical reverse-phase HPLC
Proteolysis experiments Limited proteolysis experiments with thermolysin were con-ducted on apoNgb at 25C with the proteins dissolved (0.5 mgÆmL)1) in 50 mm Tris-HCl, 0.1 m NaCl, 1 mm CaCl2, pH 7.0, using an enzyme to substrate ratio of
1 : 100 (by weight) At time intervals, aliquots were taken from the reaction mixture and proteolysis was stopped by the acidification of the solutions by adding TFA (final