Biophysical Characterization of the DNA Binding Domain of gpNu1, a
Viral DNA Packaging Protein1
David L. Bain‡, Nancy Berton‡, Marcos Ortega§, Jennifer Baran‡, Qin Yang‡, and Carlos
Enrique Catalano*,‡,||
‡Department of Pharmaceutical Sciences, School of Pharmacy, §Department
Biochemistry and Molecular Genetics, and the ||Molecular Biology Program, School of
Medicine, University of Colorado Health Sciences Center, Denver, CO 80262
*Address correspondence to this author. University of Colorado Health Sciences
Center, School of Pharmacy, 4200 East Ninth Ave, Campus Box C238, Denver, CO
80262. Office: (303) 315-8561. Fax: (303) 315-6281. E-mail:
Running Title: Biophysical Characterization of the gpNu1 DNA Binding Domain.
1 This work was supported by National Science Foundation Grant #MCB-9728550
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on February 27, 2001 as Manuscript M100517200 by guest on A
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SUMMARY
Terminase enzymes are common to double-stranded DNA viruses. These enzymes
“package” the viral genome into a pre-formed capsid. Terminase from bacteriophage λ
is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the
expression and biochemical characterization of gpNu1∆K100, a construct comprising
the N-terminal 100 amino acids of gpNu1 (Yang, et al. (1999) Biochemistry 38, 465-
477). Here we present a biophysical characterization of this construct. Thermally-
induced loss of secondary and tertiary structures is fully reversible. Surprisingly, while
loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative.
NMR and limited proteolysis data suggest that �������������� ����������������
solvent exposed and highly flexible. We therefore constructed gpNu1∆E68, a protein
consisting of the N-terminal 68 residues of gpNu1. GpNu1∆E68 is a dimer with no
evidence of dissociation or further aggregation. Thermally-induced unfolding of
gpNu1∆E68 is reversible, with concomitant loss of both secondary and tertiary structure.
The melting temperature increases with increasing protein concentration, suggesting
that dimerization and folding are, at least in part, coupled. The data suggest that
gpNu1∆E68 represents the minimal DNA binding domain of gpNu1. We further suggest
that the C-terminal ���������������������∆K100 adopt a pseudo-stable α-helix that
extends from the folded core of the protein. A model describing the role of this helix in
the assembly of the packaging apparatus is discussed.
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Introduction
Terminase enzymes are common to many of the double-stranded (ds) DNA2
bacteriophage and eukaryotic DNA viruses such as adenovirus and the herpesvirus
groups (2-4). These enzymes function to insert a viral genome into the confines of a
preformed, empty capsid. The terminase enzyme from bacteriophage λ is composed of
two virally encoded proteins, gpNu1 (181 amino acids) and gpA (640 amino acids), in a
gpA1•gpNu12 holoenzyme complex (5,6). Terminase holoenzyme possesses site-
specific nuclease (6-9), ATPase (10-13), DNA strand separation (9,14), and DNA
translocase (15,16) catalytic activities that work in concert to package viral DNA. All of
the terminase enzymes characterized to date possess a similar holoenzyme
composition (small and large subunits) and catalytic activities (5,17-21).
Replication of λ DNA proceeds through a rolling circle mechanism that gives rise to
linear concatemers of the viral genome linked in a head to tail fashion (22,23).
Packaging of viral DNA requires the excision of an individual genome from the
concatemer, and packaging of the 48.5 kb duplex within the capsid. Genome
packaging by λ terminase has been described in detail (21,24-27) and is summarized
here. Packaging initiates with the assembly of the holoenzyme at a cos site in the
concatemer. This site represents the junction between the left and right ends of
individual genomes within the concatemer (Figure 1A). Site-specific assembly at cos is
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mediated by cooperative gpNu1 binding to three repeated R-elements in the cosB
subsite of cos. Assembly of gpNu1 at cosB promotes the assembly of a gpA dimer
symmetrically disposed at cosN, yielding a stable pre-nicking complex. Site-specific
nicking of the duplex at cosN, followed by an ATP-dependent separation of the nicked
strands yields complex I, the next stable intermediate. This nucleoprotein complex next
binds an empty capsid, which triggers the transition to a mobile, ATP-driven
translocation complex that inserts DNA into the capsid. Upon arrival at the next
downstream cos site, terminase again nicks the duplex, and strand separation results in
release of the DNA-filled capsid and re-generation of complex I.
The gpA subunit of λ terminase appears to possess all of the catalytic activities required
for genome packaging, but the efficiency of each reaction is strongly stimulated by the
smaller gpNu1 subunit (7,8,12-14,16,28). Moreover, gpNu1 is required for specific and
high-affinity gpA DNA binding interactions (29), and likely contributes to the exceptional
stability of the pre-nicking complex and complex I (30,31).
Our lab is interested in the biochemical and biophysical mechanisms of DNA packaging
by phage λ terminase. Central to the packaging process is the cooperative assembly of
gpNu1 and gpA at cos (Figure 1A). To define this assembly process at a molecular
level requires an understanding of the structural features governing physical interaction
between the enzyme subunits and with DNA. Towards this end, we have sought to
2 Abbreviations Used. β-ME, 2-mercaptoethanol; cos, cohesive end site, the junction between individual genomes in immature concatemeric λ DNA; dsDNA, double-stranded DNA; gpA, the large subunit of phage λ terminase; gpNu1, the small subunit of phage λ terminase; gpNu1¨(��� D JS1X� FRQVWUXFW WUXQFDWHG DW *OX-68; gpNu1¨.���� D JS1X� FRQVWUXFWtruncated at Lys100; gpNu1¨3���� D JS1X� FRQVWUXFW WUXQFDWHG DW 3UR���� ,+)� E. coli integration host factor; kb, kilobase; kDa, kilodalton; PAGE, polyacrylamide gel electrophoresis.
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define the properties governing intrinsic and cooperative DNA binding by gpNu1.
Unfortunately, the isolated gpNu1 subunit shows a strong tendency to aggregate upon
concentration (9,28,32-34), a feature that has hampered structural and biophysical
characterization of the protein.
We recently described the construction and characterization of two deletion mutants of
gpNu1: gpNu1������(35) and gpNu1������(1), proteins truncated at Pro141 and
Lys100 of full-length gpNu1, respectively. Studies of these constructs led to a model
where the C-terminal 40 residues of the protein are required for interactions with the
gpA subunit to form a catalytically-competent holoenzyme complex (Figure 1B).
Residues 100-140 promote self-association interactions that mediate cooperative DNA
binding. The N-terminal 100 residues of the protein represented by gpNu1������
contain the putative helix-turn-helix DNA binding motif postulated to play a direct role in
DNA binding. Indeed, the construct is folded in solution and binds cos-containing DNA
with reasonable specificity (1). Preliminary NMR experiments suggested that
gpNu1�������������������������������������������(1).
Here we present a detailed biophysical characterization of gpNu1����������������������
suggest that the construct consists of a functional N-terminal domain that possess a
pseudo-stable C-terminal helix extending from the folded core of the protein. We further
describe the construction and characterization of a shorter construct that clearly
demonstrates the unusual biophysical characteristics of gpNu1������������� ��������
extended C-terminal helix. The biological significance of these results is discussed.
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EXPERIMENTAL PROCEDURES
Materials and Methods. Tryptone, yeast extract, and agar were purchased from DIFCO.
Restriction enzymes were purchased from Promega. DEAE-sepharose FF and SP-
sepharose FF chromatography resins were purchased from Pharmacia. Restriction
enzymes were purchased from Promega. Guanidinium hydrochloride was purchased
from Mallinckrodt. All other materials were of the highest quality commercially available.
Bacterial cultures were grown in shaker flasks utilizing a New Brunswick Scientific
series 25 incubator-shaker. All protein purifications utilized a Pharmacia FPLC system
that consisted of two P500 pumps, a GP250-plus controller, a V7 injector, and a Uvicord
SII variable wavelength detector. UV-VIS absorbance spectra were recorded on a
Hewlett-Packard HP8452A spectrophotometer. Fluorescence spectra were recorded at
room temperature on a PTI QuantaMaster spectrofluorometer. A protein concentration
of 10 µg/ml in 10 mM potassium phosphate buffer, pH 7.4, was used and a buffer blank
was subtracted from the fluorescence spectrum. Circular dichroism (CD) spectra were
recorded on an Aviv model 62DS circular dichroism spectropolarimeter equipped with a
Brinkmann Lauda RM6 circulating water bath and a thermostated cell holder. Near-UV
CD spectra utilized a protein concentration of 1 mg/ml in a 0.1 cm strain-free cuvette.
Data were typically collected between 250-350 nm at 0.5 nm intervals using a
bandwidth of 1.5 nm and a dwell time of 30 seconds. Far-UV CD spectra utilized a
protein concentration of 100 µg/ml in a 0.1 cm strain-free cuvette. Data were typically
collected from 180-260 nm at 0.5 nm intervals using a bandwidth of 1.5 nm and a dwell
time of 30 seconds. The raw spectra were converted to molar ellipticity using the
following equation:
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θ = θobs *
MRW10 *b* c
(1)
where θ is the molar ellipticity (degrees-cm2/dmol), θobs is the ellipticity recorded by the
instrument (millidegrees), MRW is the mean residue weight [formula weight divided by
the total number of residues in the protein], b is the cell path length, and c is the protein
concentration in mg/ml (36). Protein secondary MALDI-TOF mass spectra were
obtained from the University of Colorado Health Sciences Center Macromolecular
Resource Center. Automated DNA sequence analysis was performed by the University
of Colorado Cancer Center Macromolecular Resources Core facility. Both strands of
the duplex were examined to ensure the expected DNA sequence. Prediction of protein
secondary structures based upon primary sequence data was performed by the method
of Chou and Fasman, using the DNASIS program (Macintosh version 2.0). Calculation
of protein secondary structures based upon the far-UV CD data was performed using
the SELCON program (37).
Bacterial Strains, DNA Preparation, and Protein Purification. E. coli BL21(DE3) cells
were a generous gift of D. Kroll (University of Colorado Health Sciences Center,
Denver, CO). All synthetic oligonucleotides used in this study were purchased from
Gibco/BRL and were used without further purification. Plasmids pSF1 and pAFP1,
kindly provided by M. Feiss (University of Iowa, Iowa City, IA), were purified from the E.
coli cell lines C600[pSF1] and JM107[pAFP1], respectively, using Qiagen DNA prep®
columns. All of our purified proteins were homogenous as determined by SDS-PAGE
and densitometric analysis using a Molecular Dynamics laser densitometer and the
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ImageQuant® data analysis package. Unless otherwise indicated, protein
concentrations were determined spectrally using millimolar extinction coefficients (1,32).
Construction of pNu1����� A truncated Nu1 gene was amplified by PCR using pSF1
as a DNA template. This plasmid contains the wild-type Nu1 gene cloned into a
pBR322 background (38). Primers were designed such that EcoRI and BamHI
restriction sequences were present at the 5' and 3' end, respectively, of the PCR
product. The primer sequences used to amplify pNu1� !"��������� �������#������
primer: 5'-CCT CTC CCT TTC TCC GAA TTC ATG GAA GTC AAC AAA AAG C-3';
reverse primer: 5-CTT CCT GGA TTC TTA TTC TTC AAC CTC CCG GCG-3''. The
EcoRI and BamHI restriction sequences in the above primers are indicated in italics,
while the f-MET (forward primer) and stop (reverse primer) codons are shown in bold
type. Sequences complementary to the Nu1 gene are underlined. The stop codon
present in the reverse PCR primer yield, upon amplification, a truncated Nu1 gene that
expresses only the first 68 amino acids of the protein. PCR amplification, isolation of
the PCR product, and construction of the overexpression plasmid (pNu1� !"$�����
performed as described previously (1,28,35).
Expression and Purification of gpNu1�������Four liters of 2X-YT media containing 50
µg/ml ampicillin, 25 mM potassium phosphate, pH 7.5, and 5 mM glucose were
inoculated with a 40 ml overnight culture of BL21(DE3)[pNu1� !"%�����&��� ������
isolated colony. The cultures were maintained at 37'(����������)�D. of 1.0 (600 nm)
was obtained, at which point IPTG (1.2 mM) was added. The cells were maintained at
37'(� ���������������������*�������������&�������+������ ���������,���������������
indicated, all subsequent steps were performed at 0-4'(�� The cell pellet was
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resuspended in ice cold buffer A (20 mM Tris, pH 8.0, 2 mM EDTA, 7 mM β-ME, and
10% glycerol) containing 100 mM NaCl, and the cells were disrupted by sonification.
Insoluble cellular debris was removed by centrifugation (12K x g, 30 min), and solid
ammonium sulfate was added to the clarified supernatant to 50% saturation. Insoluble
protein was removed by centrifugation (12K x g, 30 min), and proteins were then
precipitated with the addition of ammonium sulfate to 90% saturation followed by
centrifugation. GpNu1� !"����� �����������-�-90% ammonium sulfate precipitated
fractions.
The ammonium sulfate pellet was taken into buffer A and, after dialysis against the
same buffer, loaded onto a DEAE-sepharose column (200 ml) also equilibrated with
buffer A. The column was developed with a salt gradient with gpNu1� !"������������
������.���(����(����� �������������/��������+�010-PAGE and the appropriate
fractions were pooled, dialyzed against buffer A, and loaded onto a SP-sepharose
column equilibrated with the same buffer. The column was developed with a salt
gradient with gpNu1� !"�������������2"���.���(����3���� ��*������ ������������
examined by SDS-PAGE, the appropriate fractions were pooled, dialyzed against buffer
A containing 20% glycerol, and stored at –80'(���3����4�����*������������������
concentrated and/or buffer exchanged using Ultrafree-15® centrifugal filter device
according to the manufacturers instructions (Millipore).
Sedimentation Equilibrium Analysis. Experiments were carried out with a Beckman XL-
A analytical ultracentrifuge equipped with a Ti-60 four-hole rotor with six-channel, 12
mm path-length centerpieces. Absorbance optics were used throughout. Three
different protein concentrations were used with ratios of 10:3:1, with the highest protein
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concentrations of 150 µM (���2���5��$���0����������������+6��������������������������
buffer and then diluted to the concentrations indicated in each experiment. Sample
volumes were 100 µl with the inert oil FC-43 used to displace samples from the base of
the cells. Samples were allowed to equilibrate at 20K, 30K, and 40K RPM. Samples
were judged to be at equilibrium by successive subtraction of scans. The density of
each buffer solution was calculated based on the salt composition and equilibrium
temperature. The partial specific volume of gpNu1� !"���������������+�������������
partial specific volumes of the individual amino acids (39). Data chosen for analysis had
an absorbance between 0.1 and 1.5 optical density units. Each data point was an
average of four scans taken every 0.001 cm. Data were selected for analysis using the
program REEDIT (generously provided by Dr. David Yphantis). Individual and
simultaneous analyses of nine channels (three concentrations at three speeds) were
carried out to resolve assembly stoichiometry. Data were analyzed using the
appropriate functions by nonlinear least-squares parameter estimation (40) to determine
the best-fit model-dependent parameters that minimize the variance. The program
NONLIN was used ((41); kindly donated by Dr. David Yphantis). Confidence intervals
(67%) correspond to approximately one standard deviation. Nonideality was not
considered, as there was no evidence for nonideal effects.
Models incorporating different assembly stoichiometries were based upon the general
equation:
Y(r) = δ + α exp[(σ(r2-ro2)] + ΣαNKN exp[Nσ(r2-ro
2)] (2)
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Where Y(r) is the absorbance at radius r, δ the baseline offset, and α the monomer
absorbance at reference radius ro. σ is the reduced molecular weight [σ = M(1-
νρ)ω2/RT], N is the stoichiometry of the reaction, and KN is the association constant of
the reaction NM<-> MN.
Thermal Stability Studies. Thermally-induced protein denaturation experiments were
performed as described previously (32,33,35). Each data set represents the average of
at least two independent experiments. The fraction of protein in the denatured state
(FD) was determined using the equation:
FD= θN - θT
θN - θD (3)
where θT is the ellipticity at temperature T, θN and θD represent the ellipticity for the
native and denatured protein, respectively. Baseline corrections were not performed in
order to demonstrate buffer-induced alterations in the pre-transition baseline slopes.
The unfolding curves were analyzed using a complex sigmoidal curve function:
FD=
(mD * T −bD ) − (mN * T −b
N )
1+TT m
m
T
+ (mN * T −bN) (4)
where (mN*T – bN) and (mD*T - bD) describe the linear portion of the pre-transition and
post-transition baselines, respectively, at temperature T, mT is the slope of curve within
the transition region, and Tm is the melting temperature for the transition. All data sets
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were fit to the above equations by non-linear regression methods using the IGOR®
graphics/analysis package (WaveMetrics, Lake Oswego, OR).
RESULTS
We have previously described the construction, expression, and biochemical
characterization of gpNu1�����*������������������ �����������������������������(1).
The construct is a dimer in the concentration range of 5 µM to 2 mM, with no evidence
for dissociation or further aggregation. Preliminary NMR experiments suggested that
the construct might be amenable to structural studies, and we therefore sought to
further characterize the physical properties of this construct.
Thermally-Induced Unfolding of gpNu1���. Thermally-induced unfolding of
gpNu1��100 secondary and tertiary structural elements is reversible, as indicated in
Figures 2A and 2B, respectively. Moreover, the loss of tertiary structure (near-UV CD
signal) is cooperative, consistent with a folded and stable construct (Figure 2C)3. Salt
and protons stabilize protein tertiary structure, as evidenced by the significant increase
in the Tm for the transition (Table 1). Despite the observed cooperative loss of tertiary
structure, thermally-induced loss of secondary structural elements (far-UV CD signal)
appears essentially non-cooperative (Figure 2C). The steep pre-transition baseline
observed in these data make it difficult to accurately calculate the Tm for this transition.
3 GpNu1¨.��� SRVVHVVHV WZR WU\SWRSKDQ �:�2 and W49) and three tyrosine (Y41, Y50, and Y84) residues. The thermal unfolding data provides no indication for multiple unfolding transitions, which would indicate local unfolding in the vicinity of these residues. We thus interpret the loss of the near UV CD signal as reflecting global unfolding of the protein. It is feasible, however, that the melting curves reflect regional vs. global unfolding of the protein.
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Nevertheless, it is clear that salt and protons similarly affect the Tm for the unfolding
transition, whether monitored in the far-UV or near-UV region of the CD spectrum
(Table 1). Interestingly, salt and pH strongly affect the pre-transition baselines obtained
in the far-UV CD melting curves, but not the near-UV CD melting curves (Table 2).
Limited Proteolysis of gpNu1���. Evaluation of the line widths and chemical shifts in
a 1H-15N correlation spectrum of gpNu1���������������������������������� �����
construct were solvent exposed and highly flexible (T. de Beer and C.E. Catalano,
unpublished). Primary sequence analysis predicts strong α-helical character in the
region spanning residues �-���������-� �������7#�������8$���9����������������������
putative helix might be partially disrupted in the gpNu1������������*���������������
unusual unfolding properties of the protein. If this were the case, limited proteolysis of
gpNu1���������������/���������������������/����������� ���������/*�+����������
fully folded domain suitable for structural characterization. This was indeed correct.
Limited proteolysis of the gpNu1�������������������� �������������������+�+�������
two predominant products (data not shown). Analysis of these products by SDS-PAGE
and MALDI-TOF yielded molecular masses of �:�-�;1����������;1�*��������&��+�
Characterization of gpNu1����� Proteolysis studies, NMR spectral analysis, and
evaluation of secondary structure predictions for gpNu1�������������������������
minimal folded DNA binding domain of gpNu1 is located within the N-terminal �:��
amino acids of the protein. Based on these data, gpNu1� !"����������������
Expression and purification of this construct as described in Experimental Procedures
yielded 10 mg of homogenous protein per liter of cell growth.
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The UV absorbance spectrum of gpNu1� !"�����+���� of a purified protein that is
essentially devoid of contaminating nucleotide (A280:A260= 1.95) (42). An extinction
cooefficient of ε280= 13.9 mM-1•cm-1 was calculated for the protein using the method of
Gill and von Hippel (Table 3) (43,44). The progressive decrease in ε280 going from full-
length gpNu1, to gpNu1�����*��������� !"�������������������������� ����+��������
Phe in gpNu1�����*�������������������+����������� !"������� �����������������
of gpNu1� !"��/��bits a maximum of 335 nm using an excitation wavelength of 280 nm
(Table 3). This maximum blue-shifts to 350 nm and increases in intensity (1.6-fold) in
the presence of 6 molar guanidinium hydrochloride, consistent with denaturation of a
folded protein. Identical fluorescence changes are observed with full-length gpNu1 and
the gpNu1�������������7�������$*��������������������� �������� �����������������
is similar. Importantly, gpNu1∆E68 binds cos-containing DNA with an affinity essentially
identical to that of gpNu1∆K100 (T. de Beer and C.E. Catalano, in preparation).
The far-UV CD spectrum of gpNu1� !"��������������������������������������
secondary structural elements (Figure 3A). Deconvolution analysis of the spectrum is
consistent with a protein containing 30% α-helical structure as well as 22% of the
residues being in a β-sheet conformation. This represents a significant loss of α-helical
character compared to that observed in the gpNu1�������������7�������$���0�����
signals are also observed in the near-UV CD spectrum of the construct (Figure 3B),
demonstrating that the protein also possesses significant tertiary structure. The near-
UV CD spectrum of gpNu1� !"����4�������������������� �����������*����������
exception of decreased signal intensity at 276 nm and 282 nm (compare Figures 2B
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and 3B). These bands likely represent the 1Lb transitions of Tyr84, which has been
deleted in the gpNu1� !"��������
Stoichiometry of the Self-Assembly Reaction. The assembly-state of gpNu1� !"�����
determined by sedimentation equilibrium methods. Least-squares analysis of a
representative data set is presented in Figure 4 and Table 4. The data consist of three
different loading concentrations at three different rotor speeds as described in
Experimental Procedures. Each individual data set was fit to a single-species monomer
model in order to resolve an average molecular weight. The apparent molecular weight
is constant and twice that of the calculated monomer molecular weight of 7,803 Da,
indicating that the construct is largely dimeric at all concentrations examined. Further,
there is no evidence of a concentration dependent increase in molecular weight,
indicating that gpNu1� !"�������������������+������4�����������������������������
or higher order assemblies. A more rigorous analysis by simultaneous fitting of all nine
data sets to a single-species model resolved an apparent molecular weight of 17,665 ±
749 Da, 2.26-fold greater than the calculated monomer molecular weight. The quality of
the simultaneous analysis can be seen in Figure 4, with all data being well described by
the model. The square root of the variance for the simultaneous analysis (0.010 optical
density units) is also comparable to that of any individual fit, indicative of no systematic
deviations. Fitting of the data to more complex assembly schemes either resulted in no
improvement of the fit, or failure to converge. Taken together, the data demonstrate
that gpNu1� !"�����/����&��+��������&����������������������� ��-����-��<.���
Identical results were obtained in the absence or presence of 150 mM NaCl, and at
temperatures between 4'(������:'(�7�������$�
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Thermally-Induced Unfolding of gpNu1����. Thermally-induced loss of gpNu1� !"�
secondary and tertiary structure is fully reversible (Figures 3A and 3B). The loss of
tertiary structure is cooperative (Figure 3C), and possesses a Tm similar to that
observed for the gpNu1��������truct (compare Tables 1 and 5). Moreover, salt and
protons similarly affect the thermal stability of both constructs. Thermally-induced loss
of gpNu1� !"�������+������������������������&�*������������-transition baseline
is comparatively insensitive to temperature (compare Figures 2C and 3C). Moreover,
the pre-transition baseline for gpNu1� !"��� ������������ ������+���������=�7���
shown). A significant increase in Tm is observed with increasing gpNu1� !"�
concentration (Table 6). Essentially identical results are observed with gpNu1������
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DISCUSSION
GpNu1, the small terminase subunit, is responsible for site-specific assembly of a
holoenzyme complex required for genome packaging. The protein is further responsible
for the exceptional stability of multiple nucleoprotein intermediates along the packaging
pathway. Insolubility of the isolated subunit has hampered biochemical analysis of the
protein. We have previously described the construction and biochemical
characterization of gpNu1�����*�a deletion mutant of gpNu1 that comprises the N-
terminal 100 amino acids of the protein. This construct is fully soluble at concentrations
up to 15 mg/ml (1), which allows biophysical and structural studies that are not possible
with the full-length protein. Importantly, gpNu1�����������������������&������/-turn-
helix DNA binding motif found in full-length gpNu1 (see Figure 1B) (45). We therefore
initiated NMR structural studies on this construct to understand the structural basis for
the stability of gpNu1•DNA complexes.
Fluorescence, CD, and solution NMR studies demonstrated significant secondary and
tertiary structure in the construct (1). Thermally-induced unfolding of gpNu1������
reveals an unusual situation where secondary structure is lost in a non-cooperative
manner, while global folding of the protein (tertiary structure) is lost cooperatively.
Based on a variety of data, we reasoned that gpNu1��������������� ��������������
folded N-terminal domain that possesses a C-terminal helix extending from the folded
core of the protein (Figure 5). Specifically, the C-terminal 30 amino acids of
gpNu1������������������-stable helix that “unravels” in response to elevated
temperatures, much like an isolated helical peptide. This non-cooperative loss of
secondary structure is reflected in the steep pre-transition baseline observed in the far-
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UV CD melting curves. Ultimately, the folded N-terminal region of the protein
denatures, yielding the cooperative unfolding curves observed in the near-UV CD
unfolding transition.
Limited proteolysis experiments indeed demonstrate the presence of a protease-
sensitive C-terminus, and the shorter gpNu1� !"�����������������������
Fluorescence, CD, and NMR data confirm that this construct is folded in solution.
GpNu1� !"��������������������������������������/��������������������/������*�
indicating that the self-association domain of full-length gpNu1 (Lys100-Pro141) is
distinct from the dimerization region of the protein. Our data do not allow the calculation
of an equilibrium constant for protein dimerization, but suggest an upper limit in the
nanomolar range. Of interest is the observation that thermally-induced unfolding of both
constructs is concentration dependent, suggesting that unfolding and dimerization are,
at least in part, coupled.
Thermally-induced loss of gpNu1� !"� ������+� ��������� ��� ������&�*� ����;�� �����
observed for gpNu1��������.��&��*������� !"��� �������������������������-UV
CD mirrors that obtained in the far-UV CD, suggesting that loss of secondary structure
is concomitant with global unfolding of the protein. This is consistent with the
suggestion that the strongly sloping baselines in gpNu1�������� ������ ��&��� �������
from loss of secondary structure as the pseudo-stable helix unwinds. Deletion of this
region results in cooperative secondary structure loss as the entire protein unfolds.
Our data suggest that the tertiary fold of the N-terminal 68 residues in both gpNu1� !"�
and gpNu1�������������������+������������#�����+*��������������+��������-induced
denaturation yields identical fluorescence changes in both constructs. Secondly, the
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near-UV CD spectra confirm the loss of tyrosine 85 from gpNu1� !"*��������������
indicate similar folded structures for the two constructs. Finally, thermally-induced
unfolding of tertiary structure yields comparable Tm’s for both constructs, and the salt
and pH effects are virtually identical. These data suggest that deletion of residues 69 to
100 from gpNu1����������������������+�� ������� ����������������+� �����N-
terminal 68 residues of the protein. Consistently, the affinity of both constructs for cos-
containing DNA is essentially identical.
The data are consistent with the model presented in Figure 5 that describes
gpNu1� !"������ ����*�������*����� ������� DNA binding domain of gpNu1. The
model further suggests that the C-terminal 32 amino acids of gpNu1�������������
pseudo-stable helix that extends from the folded core of the protein. This is consistent
with primary sequence analysis that predicts strong α-helical character extending from
residue �-���������1�3����������������������������-��������������� -association
domain of full-length gpNu1. Truncation of the helix at Glu100 yields a disrupted helix
that is marginally stable.
Of what functional significance is this helical region of gpNu1? The proposed structural
organization of gpNu1 finds similarity to that observed in the α subunit of E. coli RNA
polymerase (46). This protein consists of two independently folded domains connected
by a flexible sequence that possess α-helical character. The N-terminal domain (NTD)
forms a homodimer that is necessary and sufficient for core enzyme assembly and site-
specific DNA binding. The C-terminal domain (CTD) is responsible for trans-activation
by a number of transcription factors. The intervening flexible helix allows the CTDs to
act as independent motional units capable of interacting with a variety of protein signals,
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while the NTD remains site-specifically bound at the promoter. A similar structural
organization is observed in the integrase enzyme from HIV. The dimeric core domains
are connected to the C-terminal DNA binding domains by a 26 residue α-helix (47). In
this case, the interdomain helix likely plays a functional role that permits dynamic
interaction of the CTDs during the integration of viral DNA.
We suggest a similar role for an intervening helix in gpNu1. The N-terminal domain
forms a homodimer that binds specifically to cos-containing DNA. Cooperative DNA
binding is driven by self-association interactions mediated by the hydrophobic domain of
the protein. Additionally, the C-terminal gpA-interactive domain of gpNu1 must promote
gpA assembly at cosN. A helical linker between these domains would allow sufficient
flexibility such that each of these contacts may be formed appropriately, simultaneously
assembling gpNu1 and gpA at cosB and cosN, respectively. Structural, kinetic, and
biophysical studies currently underway in our laboratory are directed towards a
mechanistic description of the role of this helix in the functioning of the protein, and virus
assembly.
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Orlanco, FL
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38. Feiss, M., Siegele, D. A., Rudolph, C. F., and Frackman, M. (1982) Gene 17, 123-
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Biophys. J. 36, 575-588
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Biochemical Research, Oxford University Press, New York, N.Y.
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44. Gill, S. C., and von Hippel, P. H. (1990) Anal. Biochem. 189, 283
45. Kypr, J., and Mrazek, J. (1986) J. Mol. Biol. 191, 139-140
46. Fujita, N., Endo, S., and Ishihama, A. (2000) Biochemistry 39, 6243-6249
47. Chen, J. C.-H., Krucinski, J., Miercke, L. J. W., Finer-Moore, J. S., Tang, A. H.,
Leavitt, A. D., and Stroud, R. M. (2000) Proc. Natl. Acad. Sci., USA 97(15), 8233-
8238
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ACKNOWLEDGEMENTS. The authors are indebted to Dr. Mark Manning for use of the CD
spectrometer used in this study.
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FIGURE LEGENDS
Figure 1. Panel A. Model for Terminase Assembly at cos. Details are presented in the
text. Panel B. Domain Organization and Predicted Secondary Structure of gpNu1.
Functional domains are indicated in the upper box. The helix-turn-helix motif (Lys5-
Glu24) identified by primary sequence analysis is also indicated. Secondary structural
elements were predicted by the method of Chou and Fasman.
Figure 2. Panel A. Far-UV CD Spectra of gpNu1∆K100 before (�) and after (x)
heating to 85oC for 15 minutes. Panel B. Near-UV CD Spectra of gpNu1∆K100 before
(�) and after (x) heating to 85oC for 15 minutes. The spectra presented in panels A
and B were recorded at 4�����Panel C. Thermally-Induced Unfolding of gpNu1∆K100.
Unfolding was monitored by far-UV (�) and near-UV (�) CD signals as described in
Experimental Procedures.
Figure 3. Panel A. Far-UV CD Spectra of gpNu1∆E68 before (�) and after (x) heating
to 85oC for 15 minutes. Panel B. Near-UV CD Spectra of gpNu1∆E68 before (�) and
after (x) heating to 85oC for 15 minutes. The spectra presented in panels A and B were
recorded at 4�����Panel C. Thermally-Induced Unfolding of gpNu1∆E68. Unfolding was
monitored by far-UV (�) and near-UV (�) CD signals as described in Experimental
Procedures.
Figure 4. Sedimentation Equilibrium Analysis of gpNu1∆E68. Data sets represent initial
loading concentration of 150 µM (left), 45 µM (center), and 15 µM (right) in 10 mM
sodium phosphate buffer, pH 7.0, containing 150 mM NaCl. The symbols represent
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absorbance of gpNu1������� ����������������������������������), 30,000 (▲), and
40,000 (�) rpm, respectively. For clarity, only every fifth data point is shown. The data
were acquired at 4���������� ����������������������� ��� �!����������"#��� ���
lines represent the best-fit to a single species model obtained from simultaneous
analysis of all nine data sets.
Figure 5. Model for the Structural Organization of the Terminase gpNu1 Subunit. HTH
indicates the putative helix-turn-helix DNA binding motif.
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TABLES
TABLE 1. SALT AND PROTONS AFFECT THE THERMAL STABILITY OF GPNU1∆K100
pH [NaCl] Tm (Celsius) (Tertiary Structure, near-UV CD)
Tm (Celsius) (Secondary Structure, far-UV CD)
7.2 0 52.2 ± 0.4 47.3 ± 0.7
7.2 150 mM ND* 52.7 ± 0.8
7.2 500 mM 70.2 ± 1.6 66.2 ± 2.8
8.0 0 45.7 ± 1.6 44.2 ± 0.4
7.2 0 52.2 ± 0.4 47.3 ± 0.7
6.0 0 62.0 ± 1.5 55.9 ± 0.7
*ND, not done.
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TABLE 2. SALT AND PROTONS AFFECT THE PRE-TRANSITION BASELINE FOR THERMALLY-
INDUCED GPNU1∆K100 SECONDARY STRUCTURE LOSS.
pH [NaCl] Pre-Transition Baseline (deg-1 x 103)
(Tertiary Structure, near-UV CD)
Pre-Transition Baseline (deg-1 x 103)
(Secondary Structure, far-UV CD)
7.2 0 6.0 ± 0.3 16.6 ± 0.3
7.2 150 mM ND* 10.0 ± 0.4
7.2 500 mM 6.0 ± 0.5 8.6 ± 0.4
8.0 0 7.0 ± 0.6 10.4 ± 0.4
7.2 0 6.0 ± 0.3 16.6 ± 0.3
6.0 0 6.0 ± 0.7 7.8 ± 0.3
*ND, not done.
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Table 3. Spectral Properties of the gpNu1 Constructs.
Construct Absorbance (ε280, mM-1•cm-1)
Fluorescence (Native / Denatured) (λmax, λex=280 nm)
α-Helical Content
gpNu1-FL 17.91 336 nm / 350 nm2 53%3
gpNu1�$%�� 15.24 336 nm / 349 nm4 50%4
gpNu1���� 13.9 335nm / 350 nm 30%
All absorbance and fluorescence data were obtained 10 mM potassium phosphate
buffer, pH 7.4. 1Taken from (32). 2C.E. Catalano, unpublished. 3J.D. Meyer and C.E.
Catalano, unpublished. 4Taken from (1). α-Helical content was obtained by
deconvolution of the far-UV CD spectra as described in Experimental Procedures.
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Table 4. SEDIMENTATION EQUILIBRIUM ANALYSIS OF GPNU1�����
[gpNu1����� Rotor Speed (rpm)
Apparent Molecular Weight
Mapp
Mmono
1
S2
15 µM 20,000 16,778 ± 1,321 2.15 0.0024
15 µM 30,000 17,152 ± 532 2.20 0.0030
15 µM 40,000 16,935 ± 434 2.17 0.0046
45 µM 20,000 18,217 ± 1,341 2.34 0.0074
45 µM 30,000 18,000 ± 1,045 2.31 0.0102
45 µM 40,000 17,408 ± 710 2.23 0.0106
150 µM 20,000 20,247 ± 1,952 2.60 0.0106
150 µM 30,000 18,513 ± 1,755 2.38 0.0157
150 µM 40,000 16,541 ± 769 2.12 0.0120
Global Fit (9 data sets)
- 17,665 ± 749 Da 2.26 0.0106
The data presented in Figure 4 were analyzed as described in Experimental
Procedures. 1Mapp, apparent molecular weight obtained from analysis of the data;
Mmono, monomer molecular weight based on the gene sequence. 2Square root of the
variance.
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TABLE 5. SALT AND PROTONS AFFECT THE THERMAL STABILITY OF GPNU1∆E68.
pH [NaCl] Tm, Tertiary Structure (near-UV CD signal)
Tm, Secondary Structure (far-UV CD signal)
7.2 0 54.6 ± 0.4�� 49.6 ± 0.5��
7.2 150 mM 63.3 ± 0.3�� 56.6 ± 0.3��
7.2 500 mM 68.3 ± 0.4�� 64.6 ± 0.7��
8.0 0 47.5 ± 0.2�� 45.5 ± 0.5��
7.2 0 54.6±0.4�� 49.6±0.5��
6.0 0 60.2±0.3�� 57.0±0.5��
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TABLE 6. PROTEIN CONCENTRATION AFFECTS THE THERMAL STABILITY OF THE GPNU1
CONSTRUCTS.
Construct Concentration Tm, Secondary Structure (far-UV CD signal)
gpNu1�$%�� 100 µg/ml 47.3 ± 0.7��
gpNu1�$%�� 1000 µg/ml 54.7 ± 0.8��
gpNu1���� 100 µg/ml 49.6 ± 0.5��
gpNu1���� 1000 µg/ml 54.6 ± 0.4��
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FIGURES
Suggested Location of Figures.
Figure 1- After second paragraph in the Introduction
Figure 2- After second paragraph in Results
Figure 3- After sixth paragraph in Results
Figure 4- After seventh paragraph in Results
Figure 5- After second paragraph in Discussion
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CatalanoDavid L. Bain, Nancy Berton, Marcos Ortega, Jennifer Baran, Qin Yang and Carlos Enrique
packaging proteinBiophysical characterization of the DNA binding domain of gpNu1, a viral DNA
published online February 27, 2001J. Biol. Chem.
10.1074/jbc.M100517200Access the most updated version of this article at doi:
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