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\begin{document}

\title{Dynamic aspects of pressure and temperature-stabilized intermediates of
outer surface protein A}



\author[1]{Takuro Wakamoto}%
\author[1]{Soichiro Kitazawa}%
\author[2]{Tomoshi Kameda}%
\author[3]{Ryo Kitahara}%
\affil[1]{Ritsumeikan Daigaku - Biwako Kusatsu Campus}%
\affil[2]{National Institute of Advanced Industrial Science and Technology Tokyo Bay Area Center}%
\affil[3]{Ritsumeikan University - Biwako Kusatsu Campus}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Structural characterization of alternatively folded and partially
disordered protein conformations remains challenging. Outer surface
protein A (OspA) is a pivotal protein in Borrelia infection, which is
the etiological agent of Lyme disease. OspA exists in equilibrium with
intermediate conformations, in which the central and the C-terminal
regions of the protein have lower stabilities than the N-terminal. Here,
we characterize pressure- and temperature-stabilized intermediates of
OspA by nuclear magnetic resonance spectroscopy combined with
paramagnetic relaxation enhancement (PRE). We found that the C-terminal
region of the intermediate was partially disordered; however, it retains
weak specific contact with the N-terminal region, owing to a twist of
the central \selectlanguage{greek}β-\selectlanguage{english}sheet and increased flexibility in the polypeptide chain.
The disordered C-terminal region of the pressure-stabilized intermediate
was more compact than that of the temperature-stabilized form. Further,
molecular dynamics simulation demonstrated that temperature-induced
disordering of the \selectlanguage{greek}β-\selectlanguage{english}sheet was initiated at the C-terminal region and
continued through to the central region. An ensemble of simulation
snapshots qualitatively described the PRE data from the intermediate and
indicated that the intermediate structures of OspA may expose tick
receptor-binding sites more readily than does the basic folded
conformation.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Proteins in solution fluctuate between folded and unfolded
conformations, which is often related to their functions and toxic
aggregate formation.\emph{\textsuperscript{1-4}} Outer surface protein A
(OspA) is an immunogenic lipoprotein expressed by the
spirochete\emph{Borrelia} (e.g. \emph{B. burgdorferi, B. garinii, and B.
afzelii} ), the etiologic agent of Lyme disease. It consists of 21
repeated \selectlanguage{greek}β-\selectlanguage{english}strands and a short C-terminal \selectlanguage{greek}α-\selectlanguage{english}helix. OspA also contains
two globular domains, the N-terminal (\selectlanguage{greek}β\selectlanguage{english}1--\selectlanguage{greek}β\selectlanguage{english}7) and the C-terminal
(\selectlanguage{greek}β\selectlanguage{english}11--C-terminus) domains, as well as a single-layer \selectlanguage{greek}β-\selectlanguage{english}sheet (central
\selectlanguage{greek}β-\selectlanguage{english}sheet, \selectlanguage{greek}β\selectlanguage{english}8--\selectlanguage{greek}β\selectlanguage{english}10) that connects the terminal
domains.\emph{\textsuperscript{5,6}}

OspA binds to the tick receptor TROSPA when it enters the tick
gut.\emph{\textsuperscript{7}} Since the receptor-binding sites are
occluded in the interior of the C-terminal
domain,\emph{\textsuperscript{8}} their exposure is believed to be
required for receptor-binding. Koide et al employed native state
hydrogen exchange nuclear magnetic resonance (NMR) spectroscopy to
reveal that the C-terminal region, more specifically, the
\selectlanguage{greek}β\selectlanguage{english}9--C-terminus, including a portion of the single-layer \selectlanguage{greek}β-\selectlanguage{english}sheet and the
C-terminal domain, of this protein is less stable than the N-terminal
domain. This study provided the first evidence of disordered
intermediate conformations in the C-terminal regions of the
protein.\emph{\textsuperscript{9-11}} They also reported that the
intermediate became stabilized as temperature
increased.\emph{\textsuperscript{12,13}} The temperature-stabilized
intermediate was characterized using solution
NMR,\emph{\textsuperscript{12}} differential scanning
calorimetry,\emph{\textsuperscript{12,13}} and small angle X-ray
scattering.\emph{\textsuperscript{11}} Moreover, our group found that
the pressure-stabilized intermediate was nearly fully disordered in the
entire C-terminal region of the polypeptide chain, from \selectlanguage{greek}β\selectlanguage{english}9 to the
C-terminus. The formation of this intermediate was caused by exposure of
a large internal cavity in the C-terminal
domain.\emph{\textsuperscript{14}} Recently, Makabe et al reported
similar identity between a kinetic intermediate and the equilibrium
intermediate.\emph{\textsuperscript{15}} These results indicate that the
pressure- and temperature-stabilized intermediates have similar
structural characteristics to those existing under physiological
conditions.

Transition into the intermediate \emph{in vivo} may accelerate the
binding of OspA to the tick receptor, as the intermediate exposes the
receptor-binding sites (i.e. residues 236--237 and
242--244)\emph{\textsuperscript{8}} , and can interact with a receptor
distant from OspA.\emph{\textsuperscript{14}} In addition, the
intermediate may be important for efficient translocation of the protein
through the outer membrane.\emph{\textsuperscript{16}} More importantly,
OspA and its C-terminal fragment have been included as vaccine
candidates to prevent
\emph{Borrelia}transmission,\emph{\textsuperscript{17}} some of which
have been tested in Phase 3 clinical trials.\emph{\textsuperscript{18}}
A recombinant vaccine using \emph{B. burgdorferi} OspA was previously
licensed for Lyme disease (LYMErix, SmithKlineBeecham, Pittsburgh, PA,
USA) in 1998, however, the manufacturer voluntarily withdrew the product
from the market 3 years later.\emph{\textsuperscript{19}} Although human
vaccines for Lyme disease are not currently available, genetically
modified OspA, which contain protective elements from two different OspA
serotypes, continue to represent important vaccine
candidates.\emph{\textsuperscript{18}}

Although previous studies have reported that the C-terminal half of the
protein (\selectlanguage{greek}β\selectlanguage{english}9--C-terminus) exhibits lower stability than the N-terminal
half, our understanding of how the central \selectlanguage{greek}β-\selectlanguage{english}sheet and C-terminal domain
are disordered remains limited. Here, we further characterized the
pressure- and temperature-stabilized intermediates of OspA using a
paramagnetic relaxation enhancement (PRE)-assisted
NMR.\emph{\textsuperscript{20-24}} The PRE effect arises from
dipole-dipole interactions between unpaired electrons of the
paramagnetic agent and the nucleus of interest, and thus, spin
relaxation contributions show \emph{r} \textsuperscript{-6} dependence
of distance between the paramagnetic center and nucleus. Accordingly,
when the paramagnetic agent was conjugated to a portion of the central
domain, we were able to collect information on the distance between the
central \selectlanguage{greek}β-\selectlanguage{english}sheet and the N- and C-terminal domains.

\textbf{Materials and Methods}

\textbf{Sample preparation}

A soluble form of OspA,\emph{\textsuperscript{25}} consisting of
residues 18--273 whereby the membrane-associated N-terminal region
(residues 1--6) was truncated and the cysteine at residue 84 was
substituted by serine, was used as the pseudo wild-type (C84S; WT*) form
of OspA (BMRB Entry 4076). Uniformly \textsuperscript{15}N-labeled OspA
WT* and three variants (D118C, E128C, and A140C) were produced by
conventional \emph{E. coli} expression in M9 medium
with\textsuperscript{15}NH\textsubscript{4}Cl as the sole nitrogen
source. The protein was purified by a Ni\textsuperscript{2+}-affinity
column (Bio-Rad Laboratories, Hercules, CA) and a HiLoad 26/60 Superdex
75 prep grade column in the AKTA explore 10S (GE Healthcare Life
Sciences, Pittsburgh, PA). To cleave the His-tag, the protease thrombin
(GE Healthcare Life Sciences) was used. The cysteine variants were
dissolved in 20 mM Tris-HCl buffer at pH 7.0, mixed with\emph{S}
-({[}1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl{]}methyl)
methanesulfonothioate (MTSL) (Toronto Research Chemicals, Ontario,
Canada), and incubated for approximately 12 h in 20 mM Tris-HCl buffer
at 277 K. The protein solution was filtered and concentrated using a
Microcon (Merck Millipore, Billerica, MA, USA). The final protein
solution was adjusted to a concentration of 0.3 mM for PRE experiments
in 10 mM phosphate buffer (pH 5.9) containing 50 mM NaCl and
10\%\textsuperscript{2}H\textsubscript{2}O. The protein solution for
temperature experiments contained 0.4 M guanidium chloride (GdmCl) in
phosphate buffer. The spin-labeled MTSL was reduced with a 2-fold excess
of ascorbic acid relative to the protein concentration.

\textbf{2.2 NMR experiments}

High-pressure NMR experiments were performed on an AVANCE3-950
(\textsuperscript{1}H, 950.33 MHz) spectrometer (Bruker, Billerica, MA,
USA) with a cryo-probe system using a pressure-resistant NMR cell
(Daedalus Innovations, Aston, PA, USA). For temperature-variable
experiments, the slot NMR tube for high-concentration electrolyte
samples (SHIGEMI, Hachioji, Japan) was used. To measure
PRE,\textsuperscript{1}H/\textsuperscript{15}N heteronuclear
single-quantum correlation (HSQC) spectra of the MTSL-bound OspA and its
variants were collected for the oxidized and reduced forms of MTSL at
multiple pressures and temperatures. A 200 complex \selectlanguage{ngerman}× 4096 real data
matrix was acquired with spectral widths of 36.0
(\textsuperscript{15}N,\emph{F} \textsubscript{1}) and 12.98 ppm
(\textsuperscript{1}H,\emph{F} \textsubscript{2}). NMR data were
processed using Topspin version 3.2 (Bruker, Billerica, MA, USA),
NMRPipe,\emph{\textsuperscript{26}} and
NMRViewJ.\emph{\textsuperscript{27}} \textsuperscript{1}H chemical
shifts were referenced to the methyl signals of
2,2-dimethyl-2-silapentane-5-sulfonate (DSS), and \textsuperscript{15}N
chemical shifts were indirectly referenced to DSS (0 ppm
for\textsuperscript{1}H).

\textbf{2.3 Thermodynamic analysis}

The pressure-induced conformational transition from the native state (N)
to the intermediate state (I), was determined by examining changes in
NMR peak intensities, i.e. peak-area of L109 and V199 methyl groups to
estimate an equilibrium constant \emph{K} (= {[}\emph{I} {]}/{[}\emph{N}
{]}). Assuming two-state exchange between N and I, the Gibbs free energy
difference at any given pressure \selectlanguage{greek}Δ\selectlanguage{english}\emph{G} \textsuperscript{p} was
obtained according to Equation 1. The Gibbs free energy difference
\selectlanguage{greek}Δ\selectlanguage{english}\emph{G} \textsuperscript{0} and the partial molar volume difference
\selectlanguage{greek}Δ\selectlanguage{english}\emph{V} \textsuperscript{0} between the two conformers at 0.1 MPa was
estimated by assuming zero compressibility difference between the states
as follows:

\({G}^{p}=-RTln\ K={G}^{0}+{V}^{0}\left(p-p^{0}\right)\) (1)

where \emph{R} is the gas constant, \emph{T} is the absolute
temperature, \emph{p} and \emph{p} \textsuperscript{0} are high pressure
and 0.1 MPa, respectively.

\textbf{2.4 Analysis of PRE}

The ratio of peak height of a paramagnetic sample to that of a
diamagnetic sample, that is,\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia}, can be expressed as in Equation
2:\emph{\textsuperscript{22}}

\(\frac{I_{\text{para}}}{I_{\text{dia}}}=\frac{R_{2int}exp(-R_{2PRE}t)}{R_{2int}+R_{2PRE}}\)(2)

where \emph{R} \textsubscript{2int} and \emph{R}
\textsubscript{2PRE}represent the intrinsic transverse relaxation rate
and PRE effects on the transverse relaxation rate for each amide proton,
respectively; and\emph{t} is the total INEPT evolution time of the HSQC
(11.5 ms). The\emph{R} \textsubscript{2int} for each amide proton was
calculated from the half-height line-width of peaks of the diamagnetic
sample. Using Equation 2, the \emph{R} \textsubscript{2PRE} was
estimated by assigning the \emph{t} and\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} values. The\emph{R}
\textsubscript{2PRE} was then converted into distance using Equation 3:

\(r^{6}=\frac{K}{R_{2PRE}}\left(4\tau_{c}+\frac{3\tau_{c}}{1+\varpi_{H}^{2}\tau_{c}^{2}}\right)\)(3)

where \emph{r} is the distance between the unpaired electron and
nucleus, \selectlanguage{greek}\emph{ω} \selectlanguage{english}\textsubscript{H} is the Larmor frequency of
protons,\emph{K} is 1.23 \selectlanguage{ngerman}×10\textsuperscript{-32}
cm\textsuperscript{6}s\textsuperscript{-2},\emph{\textsuperscript{28}}
and\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{c} is the correlation time given as
1/\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{c} =1/\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{r}+1/\selectlanguage{greek}\emph{τ}
\selectlanguage{english}\textsubscript{e} (\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{r} is the rotational
correlation time of the electron--nucleus vector and\selectlanguage{greek}\emph{τ}
\selectlanguage{english}\textsubscript{e} is the electron spin relaxation time). Since\selectlanguage{greek}\emph{τ}
\selectlanguage{english}\textsubscript{e} (\textgreater{} 10\textsuperscript{-7} s) of the
unpaired electron is much longer than \selectlanguage{greek}\emph{τ}
\selectlanguage{english}\textsubscript{r},\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{c} is approximately equal
to\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{r}. The \selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{c} value is
approximately 12 ns for the folded conformation of OspA at 318 K and 0.1
MPa.\emph{\textsuperscript{29}} The half-height line-widths
(\selectlanguage{greek}Δ\selectlanguage{english}\selectlanguage{greek}\emph{ν}\selectlanguage{english}(hertz) = \emph{R} \textsubscript{2int}/\selectlanguage{greek}π\selectlanguage{english}) were approximately
20 Hz for amide proton signals in the protein. The distances from
the\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} were
estimated with a \selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{r} of 12 ns and\emph{R}
\textsubscript{2int} (20 Hz). Back-calculation of\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} for structural models
of OspA was performed with \selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{r} = 12 ns
and\emph{R} \textsubscript{2int} = 20 Hz.

Alternatively, estimation of PRE for the unfolded polypeptide chain is
not as straightforward due to the fluctuations in both the orientation
of the spins and its distance.\emph{\textsuperscript{30}} Therefore, the
distance \emph{r} was characterized according to the probability
distribution. Here, to simulate PREs of the OspA temperature-stabilized
intermediate, we used the simplest restraining model, in which
\emph{r}is between the distance of closest approach for the two spheres
(i.e. unpaired electron and nucleus). The \emph{r} \textsuperscript{6}
in Eq.3 can be substituted with\emph{d}
\textsubscript{0}\textsuperscript{3}\emph{L\textsuperscript{3}} ,
assuming that the time scale of the distance fluctuation is slow on the
time scale of \selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{rot}.\emph{\textsuperscript{30}d}
\textsubscript{0} and \emph{L} are the distance of the minimum and the
maximum separation, respectively. Further, the probability distribution
of \emph{r} was obtained from a trajectory of molecular dynamics
simulation for heat unfolding of the protein (see\emph{Molecular
dynamics simulation} ), and \emph{d} \textsubscript{0} and\emph{L} were
estimated from the distance distribution. The\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{c}
of 12 ns, which is for native state OspA, was used for the intermediate
conformations of the protein. More realistic restraining models were
discussed in the literature.\emph{\textsuperscript{30}}

\textbf{2.5 Molecular dynamics simulation}

Molecular dynamics (MD) simulation of 1 \selectlanguage{greek}µ\selectlanguage{english}s was performed for OspA using
the Amber18 simulator\emph{\textsuperscript{31}} with the AMBER
ff14SBonlysc force field\emph{\textsuperscript{31}} and the generalized
born (GB) solvent model.\emph{\textsuperscript{32}} The crystal
structure of the protein (PDB:1OSP) was used for the initial
conformation. The simulation was conducted with the \emph{NVT} ensemble,
and temperature (350 K) was controlled using a Langevin thermostat with
the solvent viscosity of water to 1.0 ps\textsuperscript{-1}.
Noncovalent interactions, including electrostatic, van der Waals, and
GB, were used without cutoffs. Covalent bonds, including hydrogen atoms,
were constrained using the SHAKE method and the integration time step
was 2 fs. Snapshots were recorded every 0.1 ns.

\textbf{Results and Discussion}

\textbf{3.1 Pressure- and temperature-induced changes
in\textsuperscript{1}H-\textsuperscript{15}N HSQC spectra}

\textsuperscript{1}H/\textsuperscript{15}N HSQC spectra of the three
variants were measured with the oxidized and reduced forms of MTSL at
0.1, 200, and 250 MPa for 313 K, using an AVANCE3 950 MHz
spectrometer.\textsuperscript{1}H/\textsuperscript{15}N HSQC cross-peaks
in the variant proteins were assigned to individual amino-acid residues
with reference to the assignments of WT* OspA (BMRB code:
4076).\emph{\textsuperscript{9}} To evaluate the effects of residue
substitution and the MTSL-tag, the \textsuperscript{1}H
and\textsuperscript{15}N chemical shifts of individual cross-peaks in
the HSQC spectra were compared between the WT* and MTSL-tagged cysteine
variants (D118C, E128C, and A140C) at 313 K and 0.1 MPa. Chemical shift
changes (i.e. ((\selectlanguage{greek}Δ\selectlanguage{english}\selectlanguage{greek}\emph{δ} \selectlanguage{english}H)\textsuperscript{2} +
(\selectlanguage{greek}Δ\selectlanguage{english}\selectlanguage{greek}\emph{δ}\selectlanguage{english}N/5)\textsuperscript{2})\textsuperscript{0.5}) are shown in
Figure S1. Since chemical shift changes larger than 0.2 ppm were only
observed at residues spatially proximal to the substituted residue, they
were likely the result of local changes in magnetic shielding, and thus,
the effects of residue substitution, and the MTSL-tag on the structure
and conformational dynamics of the protein are expectedly limited.

Pressure effects on the \textsuperscript{1}H/\textsuperscript{15}N-HSQC
spectra of the E128C variant reduced form are shown in Figure 2. The
HSQC cross-peaks corresponding to the folded conformation were
well-dispersed, and the chemical shifts of the backbone amide proton and
nitrogen were similar to those of WT* (Figure S1B); thus, the folded
conformation of the variant was similar to that of WT*. Pressure-induced
changes in the intensities (i.e., volumes) of the cross-peaks present in
the 0.1 MPa spectrum (the original cross-peaks) are plotted in Figure
S2. As pressure was increased to 150 MPa, the intensities of original
cross-peaks showed minimal changes. However, several peaks corresponding
to amide groups in the central and C-terminal regions preferentially and
gradually decreased when pressure exceeded 150 MPa, after which relative
intensities reached values of approximately 0.2. Additionally, many new
peaks appeared in the central portion of the spectrum, where disordered
polypeptide chains are typically observed (Figure 2). These results
indicate that the locally disordered, intermediate conformation was
partially stabilized under high pressure and occurred within 80\% of the
total protein.

Moreover, peak intensities (i.e., volumes) relative to those at
atmospheric pressure are plotted along with the residue number at 250
MPa (Figure 3A), indicating that pressure-induced conformational changes
occur at residues \selectlanguage{greek}β\selectlanguage{english}8 to the C-terminus. A few peak also showed
mountain-like intensity profiles (initial gain followed by a decrease in
intensity with pressure) similar to those of WT* (Figure S2). As
discussed previously\emph{\textsuperscript{14}} , conformational
dynamics on the microsecond-to-millisecond time scale may correlate with
broadening and missing of the cross-peaks, even at 0.1 MPa.

When the temperature was increased from 303 to 318 K at 0.1 MPa,
preferential decreases in cross-peaks were observed for residues between
\selectlanguage{greek}β\selectlanguage{english}8 and the C-terminus (Figure 3B), and relative intensities reached
values of approximately 0.2 (Figure S3). Besides, many new peaks
appeared in the central aspect of the spectrum (Figure S4), as observed
in the pressure experiments.

Similar pressure- and temperature-induced decreases in peak intensities
were observed in the \textsuperscript{1}H NMR spectra for L109 and V199
methyl protons (Figure S5), which are located within the central \selectlanguage{greek}β-\selectlanguage{english}sheet
and C-terminal domain of the variant (data not shown). The transitions
of the two side-chain signals, occurring in the same pressure and
temperature ranges as those of backbone amides, serve as direct evidence
for the cooperative transition of the central \selectlanguage{greek}β-\selectlanguage{english}sheet and the C-terminal
domain into the intermediate state. Based on pressure-induced changes in
peak intensities of the methyl groups, the Gibbs free energy difference
(\selectlanguage{greek}Δ\selectlanguage{english}\emph{G} \textsuperscript{0}) and the partial molar volume difference
(\selectlanguage{greek}Δ\selectlanguage{english}\emph{V} \textsuperscript{0}) between the two conformers at 0.1 MPa
and 313 K were estimated to be 17 \selectlanguage{ngerman}± 3 kJ/mol and \selectlanguage{english}-80 +- 12 mL/mol,
respectively, which are slightly smaller than those of
WT*.\emph{\textsuperscript{14}} As discussed previously, the large
volume decrease observed by this transition may be the result of
collapse and hydration of the large hydrophobic cavity in the C-terminal
domain (Fig. 1).\emph{\textsuperscript{14,35}}

An interesting observation is that cross-peak intensities of several
amide groups in the N-terminal domain were relatively increased when the
central and C-terminal domains were denatured under higher pressure
(above 150 MPa) or temperature (higher than 313 K) (Figures S2 and S3).
Although we did not delve deeper into the issue, a rotational motion of
the folded N-terminal domain may have been enhanced, thereby producing
more intense signals when the central and C-terminal domains were
denatured.

\textbf{3.2 Pressure- and temperature-stabilized intermediates}

To further characterize the pressure- and temperature-stabilized
intermediates, PRE effects on the \textsuperscript{1}H-transverse
relaxation rate constant, \emph{R} \textsubscript{2} were measured for
the variant. Rather than directly measuring the relaxation rates from a
series of two-dimensional NMR experiments, we obtained the ratios of
peak heights for a paramagnetic (oxidized) sample to those of a
diamagnetic (reduced) sample, i.e.,\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} (Eq.
2).\emph{\textsuperscript{22}} Figure 4A shows the ratio for each amide
group in the E128C variant along with the residue number (i.e., 25--110)
at 0.1, and 250 MPa at 313 K. The\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} of residues from 25 to 275 are shown in Figure S6.
When the ratio was 1, no PRE effects were observed. In contrast, a lower
ratio indicated greater PRE effects. Assuming an intrinsic line width at
half-height (\selectlanguage{greek}Δ\selectlanguage{english}\selectlanguage{greek}\emph{ν}\selectlanguage{english}(hertz) = \emph{R} \textsubscript{2int}/\selectlanguage{greek}π\selectlanguage{english}) of 20
Hz and correlation time of 12 ns for the folded OspA, distances between
the paramagnetic probe and nucleus of interest were calculated by a
previously described method (Figure S7, Eq.
3).\emph{\textsuperscript{22}} Note that the multiplicity of MTSL
positions was not taken into account in the calculation. At 0.1 MPa,
ratios of less than 0.8, corresponding to a distance of
15\textasciitilde{}20 \selectlanguage{ngerman}Å from the paramagnetic probe, were observed for
residues 82, 83, 101, and 102, while values \textless{} 0.3 corresponded
to a distance of 12\textasciitilde{}15 Å for residues 103--110. These
results indicate that residues 103--110 are closer to residue 128 than
they are to residues 82, 83, 101, and 102, consistent with the structure
resolved by X-ray crystallography at atmospheric
pressure.\emph{\textsuperscript{6}} At 250 MPa,\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} showed both increased
and decreased values at several sites. For example, the ratios for
residues 82, 83, and 102 were increased by approximately 0.2, indicating
that they drifted away from residue 128. In contrast, a remarkable
decrease in the ratio for residue 59 indicated that it shifted toward
residue 128. Figure 5A shows the locations of residues 59, 62, 82, 83,
and 128, while the pressure dependence details for the ratios are shown
in Figure 5B.

Figure 4B shows the intensity ratio,\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia}, for each amide group
of the E128C variant for each residue at 303 and 318 K at 0.1 MPa. As
the temperature increased, the ratios of residues 80, 82, and 83
increased by approximately 0.1--0.2, indicating they moved away from
residue 128. Alternatively, slight decreases in the ratios for residues
33, 36, 37, 59, 62, and 105 indicated that these residues shifted closer
to residue 128. Figure 5C shows the gradual increases and decreases
in\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} of residues
59, 62, 82, and 83 as temperature increases.

Different behaviors were observed at residues 36, 37, and 106 between
increasing pressure and increasing temperature, as shown in Figures 6
and S8. As pressure increased, split cross-peaks were observed (Figure
6A). The second (new) cross-peak increased at the expense of the first
(original) cross-peak with increasing pressure. In contrast, only a
single peak was observed as the temperature was increased from 303 to
318 K (Figure 6B). These observations indicate that these amide groups
have at least two folded conformations. Assuming a two-state exchange
process, split peaks can be observed if the exchange rate,
namely\emph{k} \textsubscript{ex}, is much smaller than the chemical
shift difference (\selectlanguage{greek}Δ\selectlanguage{english}\selectlanguage{greek}\emph{ω} \selectlanguage{english}) between two states; however, a single
averaged peak is observed if \emph{k} \textsubscript{ex} is much larger
than \selectlanguage{greek}Δ\selectlanguage{english}\selectlanguage{greek}\emph{ω} . \selectlanguage{english}Figure 6C shows the\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} values of original and
new peaks for residues 36 and 37, and 106 at 200 MPa, where both
original and new peaks were observed in the HSQC spectrum. For residues
36 and 37, new peaks showed 0.2--0.4-fold smaller values than the
original peaks, indicating shorter distances in the intermediate than in
the native conformations (Figure S6). Conversely, the new peak of
residue 106 demonstrated a 0.6-fold larger value than the original peak,
indicating much longer distance in the intermediate than in the native
conformation. In the case of the averaged peak, the observed PRE effect
is the weighted population average of the PRE effects for the two
conformations, i.e., native and intermediate conformations. Since the
average \emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia}
values of residues 36, 37, and 106 at 318 K and 0.1 MPa are similar to
those of their new peaks (200 MPa and 313 K) (Figure 6D), the second
conformation might be dominantly populated at 318 K.

Pressure and temperature effects on\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} for each amide group
were also investigated for the variants D118C and A140C, as shown in
Figures S9 and S10, respectively. As pressure increased, the\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} values of residues
59--61 of the D118C variant increased by approximately 0.1--0.2 (Figure
S9A), indicating that D118 moved away from the \selectlanguage{greek}β\selectlanguage{english}3--\selectlanguage{greek}β\selectlanguage{english}4 turn in the
pressure-stabilized intermediate. As the temperature increased, the
values of residues 57--58 and 93--94 of the variant increased by
approximately 0.2--0.3 (Figure S9B), indicating that residue 118 moved
away from the \selectlanguage{greek}β\selectlanguage{english}3--\selectlanguage{greek}β\selectlanguage{english}4 and \selectlanguage{greek}β\selectlanguage{english}6--\selectlanguage{greek}β\selectlanguage{english}7 turns in the temperature-stabilized
intermediate. Similarly, as pressure or temperature increased,
the\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} values of
residues 94--97 of the A140C variant increased by 0.2--0.3, indicating
that residue 140 moved away from the \selectlanguage{greek}β\selectlanguage{english}7 in both of the intermediates
(Figure S10A and B).\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} values of residues 36, 37, and 106 for the A140C
variant are summarized in Figure S11. A pair of cross-peaks was only
observed for residue 36. Unlike E128C, the intensity ratio of the new
residue 36 peak is similar to that of the original one (Fig. S11A). The
ratios at higher temperature were significantly higher for residues 36
and 37, and slightly lower for residue 106 (Fig. S11B), showing opposite
tendencies with the data from E128C. In addition, for the variants D118C
and A140C, residues 33--39, including the \selectlanguage{greek}β\selectlanguage{english}1--\selectlanguage{greek}β\selectlanguage{english}2 turn, maintained
lower\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} values
(i.e., 0--0.6) in both intermediates.

These results indicate that the pressure- and temperature-stabilized
intermediates shared a common structural feature in which residue 128
moved away from the \selectlanguage{greek}β\selectlanguage{english}5--\selectlanguage{greek}β\selectlanguage{english}6 turn (consisting of residues 82 and 83) while
becoming more accessible to the \selectlanguage{greek}β\selectlanguage{english}3--\selectlanguage{greek}β\selectlanguage{english}4 turn region, including residues
59 and 62. Moreover, residues 118 and 140 moved away from the N-terminal
domain, yet remained accessible to the \selectlanguage{greek}β\selectlanguage{english}1--\selectlanguage{greek}β\selectlanguage{english}2 turn. As residues 118,
128, and 140 are in the turn region between \selectlanguage{greek}β\selectlanguage{english}8--\selectlanguage{greek}β\selectlanguage{english}9, \selectlanguage{greek}β\selectlanguage{english}9--\selectlanguage{greek}β\selectlanguage{english}10, and
\selectlanguage{greek}β\selectlanguage{english}10--\selectlanguage{greek}β\selectlanguage{english}11, respectively, these PRE results suggest that the central
\selectlanguage{greek}β-\selectlanguage{english}sheet, i.e., \selectlanguage{greek}β\selectlanguage{english}8--\selectlanguage{greek}β\selectlanguage{english}10, is partially disordered, and thus is located a
longer average distance from the N-terminal domain in the intermediates
than it is in the native conformation (Figure 1). Alternatively, the
\selectlanguage{greek}β-\selectlanguage{english}sheet region may retain weak specific contact with the \selectlanguage{greek}β\selectlanguage{english}1--\selectlanguage{greek}β\selectlanguage{english}2 turn,
owing to a twist of the central \selectlanguage{greek}β-\selectlanguage{english}sheet and increased flexibility of the
polypeptide chain. Such specific long-range contact has also been
observed in intrinsically disordered proteins such as
\selectlanguage{greek}α-\selectlanguage{english}synuclein.\emph{\textsuperscript{23,36}}

New peaks appeared in the central aspect of the spectrum, an area in
which disordered polypeptide chains are typically observed, as pressure
or temperature was increased. The original signals corresponding to the
N-terminal domain and the new NMR signals showed gradual decreases in
intensity after incubating the protein for a few days at 250 MPa, likely
owing to protein aggregation. Therefore, the assignment of new peaks
using the conventional triple-resonance NMR experiments has not been
completed. However, it is useful to analyze the\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} ratios for new peaks
that likely correspond to amide groups in the central and C-terminal
domains. Figure 7A shows the frequency of\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} values for all
original cross-peaks that originated from the native protein
conformation, as the paramagnetic probe was attached at residue 140 in
the central domain. The ratios were widely distributed between 0 and 1;
however, were primarily above 0.8, indicating that a folded conformation
remained near the paramagnetic probe, and many amide groups were not
accessible to the probe. Figures 7B and C show the frequencies
of\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} values of
new peaks in the pressure-stabilized and temperature-stabilized
intermediates, respectively, of the A140C variant. In the
temperature-stabilized intermediate (Figure 7C), the frequency\emph{vs.}
\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} plot had a
Gaussian shape, with a center value of approximately 0.7; frequencies of
less than 0.2 and more than 0.9 were not observed, indicating an
entirely different shape from the native conformation. Since a Gaussian
shape is typically obtained for random events, the PRE results may have
resulted from an ensemble of disordered conformations of the polypeptide
chain. The PRE provides 1/\emph{r} \textsuperscript{6}ensemble-averaged
distance information, and thus different conformations may show
different PRE effects. Even amide groups more than 25 \selectlanguage{ngerman}Å, apart from the
paramagnetic probe in the native conformation, could transiently access
the probe in the intermediates due to the increased flexibility of the
polypeptide chain, while very few amide groups showed consistently
strong PRE effects. Although the\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia}-frequency profile of the pressure-intermediate was
similar to that of the temperature, the Gaussian shape was not as
apparent, and the strong and weak \emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} ratios, i.e., \textgreater{} 0.9 and \textless{}
0.2, were still observed (Figure 7B). Since an intermediate population
is retained in both conditions (Figure S2 and Figure S3), the
differences in the\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia}-frequency profiles indicate that the
pressure-intermediate has a more compact conformation. Similar frequency
profiles were observed for the D118C and E128C variants, as shown in
Figures S12 and S13, respectively.

\textbf{3.3 Molecular dynamics simulation}

We sought to characterize the locally disordered intermediate
conformation of OspA from PRE data. However, a conversion of\emph{R}
\textsubscript{2PRE} into distance is not straightforward in this case
since the protein exists as a mixture of different conformational
states, including \textasciitilde{}80\% locally disordered,
\textasciitilde{}20\% native, and minimal amounts of completely unfolded
protein. Even for the completely unfolded state, it is not easy to
reconstruct the probability distribution of the distance between the
paramagnetic probe and each amide proton, as multiple sources of
stochastic dynamics in the system must be
considered.\emph{\textsuperscript{30}} Here, to produce potential
structural models of the intermediate conformations of WT* OspA, we
performed a 1 \selectlanguage{greek}µ\selectlanguage{english}s MD simulation with the \emph{NVT} ensemble (350 K). The
MD simulation demonstrated that temperature-induced disordering of the
\selectlanguage{greek}β-\selectlanguage{english}sheet began at the C-terminal region, followed by the central region.

Distance distributions were calculated between the paramagnetic probe
and each amide proton for four different time-regions in the MD
trajectory, corresponding to the different levels of disorder in
\selectlanguage{greek}β\selectlanguage{english}9--\selectlanguage{greek}β\selectlanguage{english}11. Figure S14 presents the distance distribution between the
C\selectlanguage{greek}\textsubscript{β} \selectlanguage{english}of residue 118/128/140 (a substitute for the
paramagnetic center) and the amide proton of residue 37 for the folded,
partially disordered, and completely disordered ensembles. A specific
simulation snapshot was obtained in the folded and partially disordered
ensembles, in which \selectlanguage{greek}β\selectlanguage{english}11, \selectlanguage{greek}β\selectlanguage{english}10, and \selectlanguage{greek}β\selectlanguage{english}9 are disordered (Figure 8A). The
distance for the closest approach \emph{d} \textsubscript{0} and that of
the maximum separation \emph{L} for the ensembles were obtained from the
distance distribution. In the partially disordered and completely
disordered ensembles, the distance distribution became wider than that
of the folded ensemble.\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} profiles of the folded and the partially disordered
ensembles, as predicted by the MD simulation, are shown in Figure 8B
(see Methods). Increasing the ratios for residues 33, 93, and 94 of the
D118C variant can be explained by the partially disordered ensemble 3
(PDE3, Figure S8). Meanwhile, the increased ratios of residues 80--83,
103, and 106 of the E128C may have resulted from partially disordered
ensembles 1 and 2 (PDE1 and PDE2). Further, PDE1 and PDE2 can explain
the incremental ratios for residues 36, 37, 94, and 95 of the A140C
variant. However, no ensemble can explain the decreases observed in the
ratios of residues 33-37, 59, and 62. In short, any single specific
ensemble cannot explain the entire profile of \emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia}. Moreover, there is no
evidence to suggest that the new peaks corresponding to the residues in
the central \selectlanguage{greek}β-\selectlanguage{english}sheet appeared in the central portion of the spectrum.
Hence, we do not deny broadening and missing of HSQC cross-peaks owing
to conformational dynamics; however, we speculate that the central
\selectlanguage{greek}β-\selectlanguage{english}sheet does not become unfolded, but rather partially disordered, and
contains heterogeneous conformations in the intermediates.

Thus, the intermediate state of the protein is likely a mixture of
conformations, with different levels of partial disorder in the central
\selectlanguage{greek}β-\selectlanguage{english}sheet. This idea is consistent with the conformational heterogeneity
of the equilibrium intermediate mapped by native state hydrogen exchange
NMR and scanning mutagenesis.\emph{\textsuperscript{10,11}} To satisfy
the entire profile more quantitatively, PRE should be measured under
conditions in which the intermediate state is more dominantly
stabilized, and larger numbers of snapshots should be generated via MD
simulation of the partially disordered protein.

\textbf{3.4 Functional significance of the intermediate conformations}

The current PRE results provide further evidence of disorder in the
central and C-terminal regions of the OspA intermediates and
similarities between the effects of pressure and temperature on the
protein. In addition, a possible ensemble of partially disordered
conformations was suggested from the MD simulation. These data indicate
that the intermediates more readily expose the tick receptor-binding
sites (i.e., residues 236--237 and 242--244)\emph{\textsuperscript{8}}
than does the basic folded conformation. Hence, the transition into the
intermediates\emph{in vivo} may accelerate the binding of OspA to the
tick receptor. Although the folded OspA protein and its C-terminal
fragment have been included as vaccine candidates to prevent
\emph{Borrelia}transmission,\emph{\textsuperscript{17}} the
intermediates, in which the central and C-terminal domain are
disordered, are unlikely to be recognized by antibodies targeted at the
folded C-terminal portion, thereby reducing the effectiveness of the
vaccine. The disordering of the C-terminal domain is also considered to
be important when the protein is translocated through the outer membrane
in the C-terminal-to-N-terminal direction. However, destabilization of
the C-terminal domain by site-specific mutation induced protein
degradation.\emph{\textsuperscript{16}} Results from the present and
previous studies indicate that a delicate interplay between localized
conformational dynamics and thermodynamic stability may be required for
OspA function.

\textbf{Conclusion}

Although structural characterization of protein intermediates remains
challenging, the partial knowledge that we gathered here not only
expands the current understanding on the molecular mechanism of protein
function, but also facilitates novel drug design. The intermediate state
of OspA consists of a mixture of conformations with different levels of
partial disorder in the C-terminal region. The C-terminal region of the
intermediate is partially disordered; it however retains weak specific
contact with the N-terminal region, owing to a twist of the central
\selectlanguage{greek}β-\selectlanguage{english}sheet and increased flexibility of the polypeptide chain. These
intermediates may escape antibody recognition. Information regarding
localized conformational dynamics and thermodynamic stability might be
useful for the rational design of effective OspA vaccines. Moreover,
since PRE observation under high pressure is only limited in ubiquitin,
the present is a confirmation of PRE under high
pressure.\emph{\textsuperscript{24}} A combination of PRE-assisted
high-pressure NMR and MD simulation is, in general, useful for
characterizing the dynamic structures of functionally essential protein
intermediates.



\textbf{ASSOCIATED CONTENT}

\textbf{Supporting Information} Figure S1-S14

\textbf{Accession codes}

OspA (\emph{Borrelia burgdorferi} ), UniProtKB-Q45040

\textbf{Author contributions}

The manuscript was written by R. K. NMR measurements and analyses were
performed by T. W., S. K., and R. K. MD simulations were performed by T.
K. All authors reviewed and approved the final manuscript.

\textbf{Notes}

The authors declare no competing interests.



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Figure legends

Figure 1. Three-dimensional structure of OspA. Residues substituted to
cysteine and with the attached spin-labeled MTSL are depicted as
spheres. OspA contains the N-terminal (\selectlanguage{greek}β\selectlanguage{english}1--\selectlanguage{greek}β\selectlanguage{english}7) and C-terminal (\selectlanguage{greek}β\selectlanguage{english}11 to
the C-terminus) globular domains, which are connected by a single-layer
\selectlanguage{greek}β-\selectlanguage{english}sheet (\selectlanguage{greek}β\selectlanguage{english}8--\selectlanguage{greek}β\selectlanguage{english}10). The image was prepared using
Chimera.\emph{\textsuperscript{33}} Internal cavities were detected
using CASTp server with 1.4 \selectlanguage{ngerman}Å radius probe.\emph{\textsuperscript{34}}

Figure 2. \textsuperscript{1}H/\textsuperscript{15}N-HSQC spectra of the
reduced OspA E128C variant form as pressure was gradually increased from
0.1 to 250 MPa at 313 K (black, 0.1 MPa; blue, 50 MPa; cyan, 100 MPa;
green, 150 MPa; purple, 200 MPa; red, 250 MPa). (Inset) Change in
chemical shifts of V199 (not visible in the main plot).

Figure 3. Resonance intensities (i.e. peak volumes) of the OspA E128C
variant at different pressures and temperature conditions. (A) Resonance
intensities at 250 MPa relative to those at 0.1 MPa (313 K) as a
function of residue number. (B) Resonance intensities at 318 K relative
to those at 303 K (0.1 MPa) as a function of residue number.

Figure 4. Intensity ratios,\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia}, for each amide group of the E128C variant for each
residue. (A) Pressure dependence of the \emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} value at 313 K. (B)
Temperature dependence of the\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} value at 0.1 MPa. Secondary structures are indicated
at the top of the panels. Error bars for \emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} were estimated from
the noise considering error propagation, i.e.\(\left(\frac{I_{\text{para}}}{I_{\text{dia}}}\right)=\left(\frac{I_{\text{para}}}{I_{\text{dia}}}\right)\sqrt{\left(\frac{I_{\text{para}}}{I_{\text{para}}}\right)^{2}+\left(\frac{I_{\text{dia}}}{I_{\text{dia}}}\right)^{2}}\).

Figure 5. Pressure and temperature dependence of peak height ratios of a
paramagnetic sample (E128C) to those of a diamagnetic sample,
i.e.,\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia}. (A)
Locations of residues 59, 62, 82, 83, and 128 in OspA. (B)
Pressure-induced changes in the ratios of residues 59, 82, and 83 at 313
K. (C) Temperature-induced changes in the ratios of residues 59, 62, 82,
and 83 at 0.1 MPa. Panel A was prepared using
Chimera.\emph{\textsuperscript{33}}

Figure 6. Pressure- and temperature-dependent effects on selected HSQC
cross-peaks. (A) Selected \textsuperscript{1}H/\textsuperscript{15}N
HSQC spectral regions for the reduced MTSL-E128C variant at multiple
pressures (black, 0.1 MPa; blue, 50 MPa; cyan, 100 MPa; green, 150 MPa;
purple, 200 MPa; red, 250 MPa). (B)
Selected\textsuperscript{1}H/\textsuperscript{15}N HSQC spectral regions
of the protein at multiple temperatures (black, 303 K; blue, 308 K;
green, 313 K; red, 318 K). Assignments for G36 and G106 peaks are
depicted at the top of the 0.1 MPa-peak in panel A or 303 K-peak in
panel B. The peaks of G36 and G106 are marked with asterisks.
(C)\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} values of
original and new peaks of residues 36, 37, and 106 at 200 MPa.
(D)\emph{I} \textsubscript{para}/\emph{I} \textsubscript{dia} values of
the averaged peaks of residues 36, 37, and 106 at 303 and 318 K.

Figure 7. Frequency of\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} values calculated from HSQC cross-peaks for the
A140C variant. (A) Frequency of\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} of original cross-peaks at 0.1 MPa and 303 K,
corresponding to the native state. (B) Frequency of \emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} of new cross-peaks at
250 MPa and 303 K, corresponding to the pressure-stabilized
intermediate. (C) Frequency of\emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia} of new cross-peaks at 0.1 MPa and 318 K,
corresponding to the temperature-stabilized intermediate. Broken lines
depict value 1.

Figure 8. \emph{I} \textsubscript{para}/\emph{I}
\textsubscript{dia}profiles on the basis of MD simulation. (A) MD
simulation snapshots of heat unfolding (snapshots after 0.2 ns, 62.7 ns,
73.1 ns, and 80.7 ns from top to bottom, respectively). (B)\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} ratios for the
conformational ensembles of different levels of disorder in \selectlanguage{greek}β\selectlanguage{english}9-\selectlanguage{greek}β\selectlanguage{english}11
(black, the folded ensemble (16 snapshots in the MD trajectory of 0.1
ns\textasciitilde{}1.6 ns); red, the partially disordered ensemble 1
(PDE1, 31 snapshots in the MD trajectory of 60 ns\textasciitilde{}63
ns); blue, the partially disordered ensemble 2 (PDE2, 121 snapshots in
the MD trajectory of 63.1 ns\textasciitilde{}75.1 ns); green, the
partially disordered ensemble 3 (PDE3, 65 snapshots in the MD trajectory
of 75.2 ns\textasciitilde{}81.6 ns)).\emph{I}
\textsubscript{para}/\emph{I} \textsubscript{dia} ratios are calculated
from the distance distribution between the C\selectlanguage{greek}\textsubscript{β}\selectlanguage{english}of residue
118 (top)/128 (middle)/140 (bottom) (a substitute for the paramagnetic
center) and the amide proton of each residue with\selectlanguage{greek}\emph{τ}
\selectlanguage{english}\textsubscript{r} of 12 ns (see Fig. S14). Panel A was prepared using
Chimera.\emph{\textsuperscript{33}}\selectlanguage{english}
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Figure 1\selectlanguage{english}
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Figure 5\selectlanguage{english}
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Figure 6\selectlanguage{english}
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Figure 7\selectlanguage{english}
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Figure 8

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