Biophysical Characterization of the DNA Binding Domain of gpNu1, a Viral DNA Packaging Protein

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:

[email protected].

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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37. Sreerama, N., and Woody, R. W. (1993) Anal. Biochem. 209, 32-44


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44. Gill, S. C., and von Hippel, P. H. (1990) Anal. Biochem. 189, 283

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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.

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Biophysical Characterization of the DNA Binding Domain of gpNu1, a Viral DNA Packaging Protein

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