Introduction
Proteins in solution fluctuate between folded and unfolded
conformations, which is often related to their functions and toxic
aggregate formation.1-4 Outer surface protein A
(OspA) is an immunogenic lipoprotein expressed by the spirocheteBorrelia (e.g. B. burgdorferi, B. garinii, and B.
afzelii ), the etiologic agent of Lyme disease. It consists of 21
repeated β-strands and a short C-terminal α-helix. OspA also contains
two globular domains, the N-terminal (β1–β7) and the C-terminal
(β11–C-terminus) domains, as well as a single-layer β-sheet (central
β-sheet, β8–β10) that connects the terminal
domains.5,6
OspA binds to the tick receptor TROSPA when it enters the tick
gut.7 Since the receptor-binding sites are
occluded in the interior of the C-terminal
domain,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
β9–C-terminus, including a portion of the single-layer β-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.9-11 They also reported that the
intermediate became stabilized as temperature
increased.12,13 The temperature-stabilized
intermediate was characterized using solution
NMR,12 differential scanning
calorimetry,12,13 and small angle X-ray
scattering.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 β9 to the
C-terminus. The formation of this intermediate was caused by exposure of
a large internal cavity in the C-terminal
domain.14 Recently, Makabe et al reported
similar identity between a kinetic intermediate and the equilibrium
intermediate.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 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)8 , and can interact with a receptor
distant from OspA.14 In addition, the
intermediate may be important for efficient translocation of the protein
through the outer membrane.16 More importantly,
OspA and its C-terminal fragment have been included as vaccine
candidates to prevent Borreliatransmission,17 some of which have been tested
in Phase 3 clinical trials.18 A recombinant
vaccine using 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.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.18
Although previous studies have reported that the C-terminal half of the
protein (β9–C-terminus) exhibits lower stability than the N-terminal
half, our understanding of how the central β-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.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 r -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 β-sheet and the N- and C-terminal domains.
Materials and Methods
Sample preparation
A soluble form of OspA,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 15N-labeled OspA
WT* and three variants (D118C, E128C, and A140C) were produced by
conventional E. coli expression in M9 medium with15NH4Cl as the sole nitrogen source.
The protein was purified by a Ni2+-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 withS -([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%2H2O. 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.