From the Institute of Virology and Immunology,
University of Würzburg, Versbacher Strasse 7, 97078 Würzburg, Germany, the ¶ Advanced Biomedical Computing
Center, SAIC/NCI-Frederick Cancer Research and Development Center,
National Institutes of Health, Frederick, Maryland 21702-1201,
Leiden University Medical Center, AZL P4-22, P.O. BOX 9600, 2300RC Leiden, The Netherlands, and the ** M. P. Chumakov Institute
of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical
Sciences, 142782 Moscow Region, Russia
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ABSTRACT |
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A cysteine proteinase, papain-like
proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the
expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage
between Gly111 and Asn112, far upstream
of its own catalytic residue Cys1054. In this report, using
bioinformatics tools, we predict that, unlike its distant cellular
homologues, HCoV PL1pro and its coronaviral relatives have a poorly
conserved Zn2+ finger connecting the left and right hand
domains of a papain-like fold. Optical emission spectrometry has been
used to confirm the presence of Zn2+ in a purified and
proteolytically active form of the HCoV PL1pro fused with the
Escherichia coli maltose-binding protein. In
denaturation/renaturation experiments using the recombinant protein,
its activity was shown to be strongly dependent upon Zn2+,
which could be partly substituted by Co2+ during
renaturation. The reconstituted, Zn2+-containing PL1pro was
not sensitive to 1,10-phenanthroline, and the Zn2+-depleted
protein was not reactivated by adding Zn2+ after
renaturation. Consistent with the proposed essential structural role of
Zn2+, PL1pro was selectively inactivated by mutations in
the Zn2+ finger, including replacements of any of four
conserved Cys residues predicted to co-ordinate Zn2+. The
unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme.
Proteolytic enzymes control a large variety of processes in
cellular organisms and viruses. Some proteases can completely digest
most proteins, while others are highly selective, cleaving only one
bond in a protein or a set of proteins (1). Mechanistically, four
classes of proteases have been recognized, and they are named in
accordance with the chemical nature of their catalytic site, namely
cysteine proteases, serine proteases, aspartate proteases, and
metalloproteases (Zn2+) (2). Each protease class can be
further divided into protein families that are united by a common
origin and a common structural fold (3). Recently, however, it has
become evident that more complex relationships exist among proteases.
Thus, proteolytic enzymes employing different catalytic mechanisms
within the framework of the same structural fold have been identified
(showing that protease families can extend across the borders
separating classes (4-6)) and proteases that are decorated with
determinants of nonproteolytic activity, for example,
polynucleotide-binding cysteine proteases (7-9) and
Zn2+-binding nonmetalloproteases (see below) have also been
described. The proteases that exhibit these unusual features are very
often encoded by positive sense RNA viruses. Many of these viruses rely upon proteolytic enzymes to regulate viral and cellular gene expression and virion morphogenesis (10-12).
The majority of RNA viral proteinases are recognized as being very
distantly related to either cellular chymotrypsin-like or papain-like
proteases (CLpro and PLpro,
respectively).1 RNA viral
CLpro enzymes have been found that exhibit three different catalytic
triads, the canonical Ser-His-Asp and two variations not found in
cellular proteins, Cys-His-Asp and Cys-His-Glu (5, 13-16). Some RNA
viral CLpro enzymes, with the canonical or Cys-His-Asp catalytic triads
(e.g. the hepatitis C virus NS3 and picornavirus 2A
proteases, respectively) employ a structural Zn2+ bound by
two loops of the two One of the groups of RNA viral PLpro enzymes includes seven ortologous
and paralogous coronaviral proteinases. Three coronaviruses (murine
hepatitis virus (MHV) (31, 32), human coronavirus 229E (HCoV) (33), and
porcine transmissible gastroenteritis virus (TGEV) (34)) encode two
divergent copies of PLpro (PL1pro and PL2pro), while avian infectious
bronchitis virus (IBV) (23, 26, 35) encodes only one PLpro. These
enzymes are produced as part of large replicative precursor proteins
(pp1a and pp1ab), with pp1ab being expressed by a mechanism involving
( Computer Sequence and Structure Analyses--
Amino acid
sequences were derived from Swiss-Prot and GenbankTM data
bases, and protein structures were derived from the Brookhaven data
base (Protein Data Bank, Brookhaven National Laboratory, Upton, NY).
Sequence alignments were produced using a family of Clustal programs
(43, 44) and the Macaw workbench (45). Nonredundant sequence data bases
were searched with single sequences (46), blocks (47), and Hidden
Markov Models trained on multiple sequence alignments (48). These
alignments were also sent as input for the PhD (49) or DSC (50)
programs to predict secondary structure. The 123D program (51) was used
for the fold prediction by threading. The structure superimposition and
three-dimensional protein homology modeling (52) were done and
evaluated with the help of the Quanta 97 and Whatif 4.99 (53) packages.
Analysis of the structure-based alignments was assisted by the CORE
package (54, 55). Ribbons 2.81 (56) was used to display protein structures, and DSSP (57) was used to calculate secondary structure.
Plasmids--
Plasmid pT7-IRES-Pap, encoding amino acids (aa)
1-1315 of pp1a/pp1ab, has been described previously (41). DNA was
amplified from pT7-IRES-Pap by standard polymerase chain reaction
procedures using oligonucleotides I
(5'-ACTGCCATGGGTGGTATTTTGGCAGTAATA-3') and II
(5'-TTATCACTTGGTAGAAAGCTACATTGTC-3'). The polymerase chain reaction
product was cloned into the XmnI site of pMal-c2 (New England Biolabs, Schwalbach, Germany), resulting in pMal-PL1, and the
construction was verified by sequencing. Plasmid pMal-PL1 encodes an
isopropyl-1-thio- Site-directed Mutagenesis--
A modified protocol of
site-directed mutagenesis (58) was used. Two partially complementary
oligonucleotides carrying the desired mutation were used in a
polymerase chain reaction with plasmid DNA as template. The parental
DNA was digested with DpnI, and the polymerase chain
reaction products were used to transform E. coli Top 10F'
cells. In vivo recombined plasmid DNA was isolated from
individual clones and sequenced. Then DNA fragments from the parental
plasmids were replaced with appropriate DNA fragments from the mutated plasmids.
Bacterial Expression and Purification of Recombinant
Proteins--
Plasmids pMal-c2 and pMal-PL1 were used to transform
E. coli TB1 cells. Single colonies were inoculated into LB
medium supplemented with 100 µM ZnOAc and incubated at
37 °C until an A600 of 0.5 was reached.
Expression of the recombinant protein was induced with 1 mM
isopropyl-1-thio- In Vitro Trans-cleavage Assay--
The proteolytic activity of
in vitro synthesized proteins expressed from pT7-IRES-Pap
and its derivatives or the activity of bacterially expressed MBP-PL1
was assayed using in vitro synthesized, [35S]Met-labeled substrate, representing aa 1-956 of
pp1a/pp1ab (41). The proteolytic reaction was done at 30 °C by
incubation of 7 µl of the reticulocyte lysate substrate with either
20 µl of in vitro synthesized enzyme for 3 h (41) or
1 µg (0.5 µl) of recombinant protein for 1 h. The reaction
products were immunoprecipitated with the polyclonal rabbit antiserum
IS1720 (specific for aa 41-250 of pp1a/pp1ab) and analyzed by
10-17.5% gradient SDS-polyacrylamide gel electrophoresis (41).
Radioactively labeled polypeptides were visualized by autoradiography.
Denaturation and Renaturation of MBP-PL1--
Two 1-ml aliquots
(I and II) of purified MBP-PL1 (1 mg/ml) were mixed with 9 volumes of
buffer A containing 8.0 M urea. After incubation at
20 °C for 4 h, aliquot I was adjusted with EDTA to a final
concentration of 10 mM, and aliquot II was adjusted with
ZnOAc to a final concentration of 300 µM. The aliquots
were subsequently dialyzed twice against 500 volumes of buffer A
containing 1 mM EDTA (aliquot I) or 100 µM
ZnOAc (aliquot II) at 4 °C for 16 h. The dialyzed material was
then concentrated 10-fold using Centricon-10 membranes (Amicon). The
EDTA-treated sample (aliquot I) was subjected to a second round of
denaturation as described above and divided into three aliquots (Ia,
Ib, and Ic). Aliquot Ia was adjusted with EDTA to a final concentration
of 10 mM, aliquot Ib was adjusted with ZnOAc to a final
concentration of 300 µM, and aliquot Ic was adjusted with
CoOAc to a final concentration of 300 µM. The aliquots
were dialyzed against 500 volumes of buffer A containing 1 mM EDTA (aliquot Ia), 100 µM ZnOAc (aliquot
Ib), or 100 µM CoOAc (aliquot Ic).
Determination of Metal Content--
The recombinant protein
solutions were analyzed for the presence of Ca2+,
Co2+, Cu2+, Fe2+, Mg2+,
Mn2+, Ni2+, Pb2+, and
Zn2+ by ICP-OES on a JY30 Plus spectrometer (Jobin Yvon,
France). Prior to analysis, all samples were treated twice with
Chelex-100 resin by mixing in suspension at 4 °C for 30 min.
Coronaviral PL Proteases Are Predicted to Have the
First, we extended previous analyses (26, 31, 33, 34) of the primary
structure of the coronavirus PL protein family using multiple sequence
comparison tools (see "Experimental Procedures"). A set of multiple
alignments of the conserved PL domains, consisting of aa 202-216, was
generated, and an overall alignment was constructed (Fig.
1, A and C). A
number of different approaches were then used to search the data bases
for similarities (see "Experimental Procedures"). Only several
matches of marginal statistical significance were detected with
cysteine proteases, namely between the coronavirus PLpros and the Lpro
of different strains of FMDV (26) and also proteases of the ubiquitin
isopeptidase T family (60). These weak similarities were structurally
reasonable, since in the matches (putative) catalytic Cys or His
residues were aligned (data not shown). This failure to identify a
pronounced similarity between coronaviral and the other PLpros was,
perhaps, not surprising given that pairwise similarity within the
coronavirus PLpro family itself is very low (13-32% identical
residues). Thus, to check for the existence of more remote structural
similarities, which were not evident at the amino acid sequence level
(61), the predicted secondary structure of the coronavirus PLpros was
threaded (62, 63) through the Protein Data Bank. Even then, no high scores were returned. However, consistent with the above observations and a previous analysis (26), papain was among the three top hits (Z
score was 3.16) with the catalytic cysteines of papain and the
coronavirus PLpros being matched (data not shown). It is also
noteworthy that coronaviral PLpros were predicted to belong to the Coronaviral PL Proteases Contain a Unique Zn2+ Finger
Connecting the Two Domains of a Papain-like Fold--
In the alignment
displayed in Fig. 1A, three structural elements of the
cellular PLpros,
When the predicted HCoV PL1pro Zn2+ finger was compared
with the structural data base of Zn2+ fingers (67, 68), the
nucleic acid binding domain of human transcriptional elongation factor
TFIIS, which has a three-stranded, antiparallel,
From the comparative sequence analyses outlined above, we predicted
that coronaviral PLpros are composed of the two domains of the
papain-like fold which, unlike their known cellular relatives, are
connected by a Zn2+ finger.
Purified PL1pro Fused with E. coli Maltose-binding Protein Is
Proteolytically Active--
To address the enzymatic and predicted
Zn2+ binding properties of coronaviral PLpros
experimentally, we decided to study a recombinant, purified HCoV PL1pro
that was amenable to genetic and biochemical manipulations. The minimal
HCoV PL1pro domain, which had been shown to be active when synthesized
in a reticulocyte lysate (41), was expressed in E. coli as
the fusion protein MBP-PL1 (Fig.
2A, lanes
2 and 3). MBP-PL1 was partly purified by amylose
affinity chromatography (Fig. 2A, lane
4) and assayed for proteolytic activity. The recombinant
MBP-PL1, but not a derivative carrying a replacement of the catalytic
Cys1054 by serine, was proteolytically active (Fig. 2B,
lanes 1 and 2). The proteolytic
activity of recombinant MBP-PL1 was identical to that of in
vitro synthesized HCoV PL1pro (Fig. 2B,
lanes 3 and 4; Ref. 41).
HCoV PL1pro Is a Zn2+-binding Enzyme--
The partly
purified MBP-PL1 was analyzed for eight metals by ICP-OES. No other
metals except Zn2+ and Fe2+ were detected in
significant amounts. The Zn2+/Fe2+ ratio was
approximately 3-4.5/1 (Table I). MBP,
expressed and purified under identical conditions, bound only
background amounts of the metal ions, confirming that it is the
coronaviral portion of the fusion protein that binds Zn2+.
It was calculated that HCoV PL1pro binds Zn2+ in equimolar
amounts.
Zn2+ Is an Essential Structural Cofactor of
Proteolytically Active HCoV PL1pro--
In a series of preliminary
experiments, we observed that if ZnOAc was not added to the bacterial
growth media, purified MBP-PL1 was proteolytically less active and
there was a decreased amount of Zn2+ and increased amount
of Fe2+ bound to the protein (data not shown). This
observation indicated that Zn2+ might be an essential
co-factor of PL1pro. To test this hypothesis, MBP-PL1 was denatured in
the presence of 8 M urea and then renatured by dialysis
against buffer A supplemented with either ZnOAc or EDTA (72). MBP-PL1
renatured in the presence of Zn2+, but not EDTA, was
proteolytically active (Fig. 3,
lanes 4 and 5). Furthermore, the
Zn2+-depleted MBP-PL1 (apoenzyme) could be reactivated
after a second round of denaturation and renaturation in the presence
of ZnOAc or, to a lesser degree, CoOAc (Fig. 3, lanes
6-8). Importantly, Fe2+ could not substitute
for Zn2+, the addition of 100 µM ZnOAc to the
apoenzyme did not restore activity, and the reconstituted
Zn2+-PL1pro was not inhibited by 10 mM
1,10-phenanthroline (data not shown). These results strongly suggest
that Zn2+ is tightly bound to the PL1pro and that
coordination of the Zn2+ has to occur during folding of the
protein. This also implies that Zn2+ plays an essential
structural rather than catalytic role.
Four Cysteine Residues Implicated in Binding Zn2+ Are
Crucial for Proteolytic Activity of HCoV PL1pro--
In our model of
the HCoV PL1pro, the Zn2+ is predicted to be tetrahedrally
coordinated by Cys1126, Cys1128,
Cys1154, and Cys1157 (Fig. 1C). If
this is correct, and in view of the strong dependence of MBP-PL1
activity on Zn2+ (Fig. 3), these four cysteine residues
should be indispensable for proteolytic activity. We tested this
prediction by assaying the proteolytic activity of in vitro
synthesized PL1pro mutants. Consistent with our prediction, mutants
with a replacement of any of the putative Zn2+-coordinating
cysteine residues were proteolytically inactive (Table
II). Furthermore, PL1pro was found to be
inactivated by two other mutations in the vicinity of the
Zn2+-coordinating Cys residues, an insertion of Val between
aa 1126 and 1127 or a deletion of Leu1155 (Table II and
Fig. 1A). The effect of the above mutations was highly
selective, since two different mutations at Cys1163, a
nonconserved residue of the Zn2+ finger, did not inactivate
the enzyme (Table II). Also, six other highly conserved residues,
Lys1048, Gly1099, Gly1102,
Val1175, Cys1203, and Asp1218, were
probed outside the Zn2+ finger. The majority of these
residues were predicted to be involved in the formation of the active
site, as can be seen from the alignment of the coronaviral and cellular
PLpros (Fig. 1A). Each of these residues (with the exception
of Lys1048) was replaced by a number of amino acids found
in the same position in other PLpro(s) or by other amino acids (Fig.
1A and Table II). In contrast to the four conserved Cys
residues of the Zn2+ finger, the majority of these residues
tolerated at least one replacement. The exception, Lys1048,
which was aligned with the oxyanion hole-forming Gln15 of
papain, did not tolerate a Glu substitution, the only tested mutation.
These results are fully compatible with our alignment of coronaviral
and cellular PLpros (Fig. 1) and indicate that the four conserved Cys
residues of the Zn2+ finger are as equally important for
the proteolytic activity of the HCoV PL1pro as the previously
identified catalytic Cys1054 and His1205
residues (41).
In this report, we describe a unique cysteine proteinase,
dependent on Zn2+ and encoded by an RNA virus. A
combination of bioinformatics, biochemical, and molecular-genetic
techniques has been used to obtain evidence that strongly supports a
structural, rather than a catalytic, role of Zn2+ in the
HCoV PL1pro. To characterize the HCoV PL1pro, we have expressed the
enzyme in E. coli as a fusion protein that was shown to be
proteolytically active in a trans-cleavage assay. The ability of PL1pro
to cleave a cognate substrate seems to be completely dependent on
additional (viral) cofactor(s), since we have been unable to develop a
peptide-based assay for this protease to date. These putative
cofactor(s) may reside in the approximately 900-aa region that
separates the catalytic domain of PL1pro and its cleavage site. This
region is known to modulate PL1pro-mediated processing in MHV (37,
73).
The most intriguing aspect of the HCoV PL1pro structure is, without
doubt, the zinc finger. We have, first of all, delineated this
structure on theoretical grounds and subsequently used a recombinant
PL1pro to characterize the Zn2+ binding properties by
spectroscopic analysis as well as the structure-function relationships
by mutational analysis. The experimental results have validated the
functional importance of the HCoV PL1pro Zn2+ finger,
although we acknowledge that it remains to be shown directly that its
four conserved Cys residues bind Zn2+. In the first
analysis, the Zn2+ finger was predicted to adopt a
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel fold (17-22). Until very recently, the
relationship between cellular and RNA viral PLpros had been primarily
based on the results of sequence (23-26) and predicted secondary
structure (27) analyses. A recent x-ray analysis of the foot-and-mouth
virus (FMDV) Lpro (leader protease), one of
the most well characterized viral PLpros (28), has proven the distant
relationship between viral and cellular PLpros by showing that the FMDV
Lpro adopts a compact version of the PLpro fold (29). Like its cellular
homologues, the FMDV Lpro employs the catalytic Cys-His dyad residues,
which are assisted by unique Asn and Asp residues replacing,
respectively, Gln forming the oxyanion hole as well as Asn
hydrogen-bonded to the catalytic His in cellular enzymes. Most
probably, similar or other replacements could be found in the catalytic
center of the other RNA viral PLpros (26) (see also below). It is
reasonable to think that the structural diversity of RNA viral
proteinases is related to the extremely high mutation rate of RNA
viruses (30). As a result, many RNA viral proteinases have diverged to
the point where sequence similarity between homologues can barely be detected.
1) ribosomal frameshifting (36). The PL1pros of MHV (37-40) and
HCoV (41) and the PLpro of IBV (42) cleave one or two sites upstream of the protease domain. HCoV PL1pro, which is the focus of the work reported here, was previously mapped between the Gly861 and
Gln1285 residues of pp1a/pp1ab. This protease releases an
N-terminal protein, p9, from the pp1a/pp1ab precursors via the cleavage
of the Gly111-Asn112 bond. The conserved
residues Cys1054 and His1205, which were
predicted to be the catalytic dyad, were found to be indispensable for
proteolytic activity of the HCoV PL1pro (41). In this paper, we predict
and provide experimental evidence to show that coronaviral PLpros
comprise a unique group of enzymes that rely upon a structural
Zn2+ within the framework of a papain-like fold.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside-inducible fusion protein, MBP-PL1, consisting of three parts: the Escherichia
coli maltose-binding protein (MBP), a linker of three artificial
aa, and the HCoV PL1pro domain consisting of aa 861-1285 of pp1a/pp1ab (the so-called "minimal" trans-active protease) (41).
-D-galactopyranoside. After incubation at 25 °C for 10 h, the cells were harvested and resuspended in 3 ml of buffer A (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM
-mercaptoethanol)/g of cell
pellet. Cells were broken with a French pressure cell (SLM Instruments
Inc.), and the lysate was clarified by centrifugation at 5000 × g for 30 min. The clarified lysate was loaded on a column containing amylose and washed with 10 column volumes of buffer A, and
MBP-containing proteins were eluted using buffer A containing 10 mM maltose. The protein concentration was determined and
adjusted to 1 or 2 mg/ml. The bacterially expressed recombinant
proteins were analyzed by SDS-polyacrylamide gel electrophoresis.
Protein preparations were stored at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
+
Structural Organization Found in Cellular Papain-like
Proteinases--
In this study, we have employed bioinformatics tools
to gain insights into the structure and function of coronaviral PLpros. The predictions derived from these analyses have then been tested experimentally. Previously, this same strategy has proven very efficient in identifying and characterizing numerous RNA viral enzymes
(for a review, see Ref. 59), including coronaviral PLpros (37,
40-42).
+
structural class, which also includes cellular PLpros. Thus, we
were encouraged to elaborate our model by producing a secondary
structure-based alignment of seven coronaviral and 11 cellular PLpros,
whose tertiary structures have been solved. This alignment includes 10 ungapped blocks encompassing the majority of the (predicted) structural
elements of the two protein sets and has interblock spacing that is
similar in the proteases of both cellular and viral origin (Fig.
1A). The interfamily amino acid conservation is most evident
in blocks II, IV, VI, and VIII, which contain both catalytic and
substrate-binding pocket residues of the cellular enzymes and four out
of eight absolutely conserved residues of the viral enzymes (three in
block II and one in block VIII). Three other invariant residues are
located between blocks V and VI and described below. The eighth
coronaviral invariant residue, Asp, is located in block IX. In an
alignment slightly different from that shown in Fig. 1A,
this residue would correspond to a highly conserved Asn, which is
hydrogen-bonded to catalytic His residue of cellular enzymes. However,
this alternative alignment was not favored by the MACAW-assisted
analysis or by the results of a site-directed mutagenesis study (see
below). In this respect, it is worth noting that the conserved Asn of
cellular PLpros does not play the catalytic role (64) and is
substituted by Lys in stem bromelain, one of the plant PLpros (65).
Importantly, in the alignment shown in Fig. 1A, the
coronavirus PL residues match the majority (77 out of 91) of cellular
PL residues with a low space variation (the so-called core residues
(55)) and only a minority (32 out of 87) of structurally less conserved
residues (Fig. 1, A and B). Taken together, all
of the above observations strongly imply that the coronaviral PLpros
may adopt a variant of the
+
fold conserved in cellular
PLpros.
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Fig. 1.
Fig. 1. Coronaviral and cellular PLpros:
structural similarities and unique features. A,
secondary structure-based sequence alignment of coronaviral and
cellular PLpros. The primary structures of HCoV PL1pro and its
coronaviral relatives (for accession numbers see C) were
aligned using ClustalW program (43) in a stepwise manner and manually
corrected with the ClustalX program (44) and the MACAW workbench (45).
The main portion of this alignment is presented as 10 ungapped blocks.
Only blocks II and III were statistically significant
(p < 10 20 and p = 1.5
13, respectively), and blocks IV and VII, excluding
MHV PL1pro, were conditionally significant, using a searching space
between blocks III and VIII and between blocks IV and VIII,
respectively. The validity of block VIII was previously confirmed by
site-directed mutagenesis of conserved His for MHV and HCoV PL1pros and
IBV PLpro (37, 41, 42). The secondary structures predicted by the PhD
program are shown at the top (SS_coronaPL; A and
a represent
-helix, and B and b
represent
-strand, predictions in capital
letters have a reliability >5 and predictions in
lowercase letters have a reliability of 5 and
less (49)). The validity of this prediction was confirmed when similar
secondary structure profiles were also returned for (i) the same
alignment using the DSC program (50) and (ii) two automatically
generated alignments containing either PL1pros or PL2pros encoded by
HCoV and TGEV (not shown). The secondary structure profile of
coronaviral PLpros was aligned with secondary structure elements
conserved in the tertiary structure of 11 cellular PLpros (SS_celPL)
(Protein Data Bank accession numbers: 1ppn, Papaya_Pap, papain (77);
1gec, Papaya_Glep, glycyl endopeptidase (78); 1ppo, caricain (79);
1yac, chymopapain (80); 1mem, cathepsin K (81); 1cjl, Human_CatL,
cathepsin L (82); 1cte, Rat_Catb, cathepsin B (83); 2aim, trypanosoma
cruzain (84); 2act, actinidin (85); 1gcb, yeast Gal6/bleomycin
hydrolase (86)). The secondary structure alignment guided a sequence
alignment of coronaviral and cellular proteases. A register of the
alignment within each block was (arbitrarily) selected to maximize
interfamily sequence similarity, although two or more poorly
discriminated alignments were produced for all blocks except blocks II
and VIII. When the three-dimensional structures of coronaviral PLpros
become available, this alignment may need to be locally adjusted. For
cellular PLpros, only a representative set of five sequences is shown.
Coloring of the alignment of 12 sequences indicates the following:
pink, invariant residues; red, residues conserved
in >50% of the sequences; green, group of similar
residues. The alignments of coronaviral and cellular PLpros highlight
the active site residues of cellular proteases (66, 78). *, principal
catalytic; +, "accessory" catalytic; 1, 2,
3, and 4, substrate-binding pocket subsites S1,
S2, S3, and S4, respectively; #, oxyanion hole-forming residue.
Beneath the alignments, a plot displaying the positional
structural variability (55) of cellular PLpros is shown.
Above the plot, the positions of conserved secondary
structure elements of cellular PLpros (66) as well as four conserved
hydrogen-forming elements consisting of one residue (not marked) in the
primary structure are displayed. Vertical axis,
space variability at a position of the alignment; horizontal
axis, numeration in the structural alignment containing only
aligned residues. B, core structural residues of the
cellular PLpros and residues conserved in cellular and coronaviral
PLpros. Using the CORE package (55), a structural alignment of 11 cellular PLpros was converted into an average PL structure. It is
characterized by the mean position of each C-
atom common in the
family. The size of the ellipsoid around each of these atoms is
proportional to the volume of atom variance. The two identical average
PL structures, consisting of 178 atoms, are displayed in the
"standard" papain orientation (66) featuring left-hand and
right-hand domains as well as the interdomain active site cleft with
the two catalytic residues of papain, Cys25 and
His159. Conserved secondary structure elements of cellular
PLpros are also marked. These structures are colored in
green and red as follows. The left structure, the
half of C-
atoms plus two atoms having the lowest space variance (91 atoms) are colored in red (core), and the remaining atoms
are in green (noncore). The right structure, 109 atoms,
whose residues were aligned with coronaviral PL residues in Fig.
1A, are shown in red (interfamily conserved
residues), and the remaining atoms are in green. Note that
the cellular PL core residues and the interfamily conserved residues
are mainly from the same pool. C, A unique Zn2+
finger connects the two domains of the PL fold of coronaviral PLpros. A
region of the coronaviral PLpros between blocks V and VI was aligned as
specified in Fig. 1A. Using the secondary structures
predicted for the PLpros (SS_coronaPL) (50) and derived from the NMR
structure (69) of the TFIIS Zn2+ ribbon (SS_TFI), an
alignment of Zn2+ fingers of coronaviral PLpros and TFIIS
was generated. The positions of these sequences in the corresponding
proteins are given on the left, and accession numbers in the
sequence data bases are shown on the right.
Coloring of the alignment is as detailed for A.
Residues involved in Zn2+ binding in TFIIS (69) are marked.
A bar depicts the region of HCoV pp1a/pp1ab characterized in
this study with the conserved blocks (Fig. 1A) shown. These
blocks are organized in three groups colored differently.
Blue, left-hand
-helix domain; green,
right-hand
-sheet domain without counterparts of
A- and
B-strands; red, Zn2+ finger domain.
Beneath the bar, the positions of the PL1pro
domain, which is conserved among coronaviruses, and the HCoV minimal
PL1pro domain determined by deletion analysis (41) are shown. The
positions of mutations (Ref. 41 and Table II) are depicted with
yellow vertical lines in the
bar and yellow amino acid background in the
alignments in A and C.
A,
B, and
-RII, which are spatially juxtaposed in front of the substrate-binding pocket and comprise a
peripheral part of the right-hand domain (Fig. 1B), were not aligned with coronaviral sequences. Two of these elements,
A and
-RII, form part of a long, poorly conserved structure found in the
middle of the cellular PLpros (66). The corresponding region between
blocks V and VI in the coronaviral PLpros was highly diverged, but,
most conspicuously, it was found to contain a unique conserved sequence
pattern characteristic of Zn2+ fingers,
CX1-2CX22-31CX1-2-[CH],
where X is any aa (Fig. 1C).
-sheet fold (zinc
ribbon) (69), was selected as being the most similar. Both proteins are
enriched with
-strands, belong to the C4 class, have a similar
spacing between the pair of the C2 halves, and have a variable spacing
separating cysteines within the C2 elements (or cysteine and histidine
in the CH element of related proteins) (Fig. 1C). The zinc
ribbon architecture is apparently conserved in a wide variety of
Zn2+ fingers (69, 70) and was shown to tolerate a large
size difference in the loop structure (71). These observations strongly
suggest that the specific features of the coronaviral PLpro
Zn2+ fingers, which are evident from the alignment shown in
Fig. 1C, may also be compatible with this architecture.
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Fig. 2.
Expression and purification of
proteolytically active HCoV PL1pro fused with the E. coli
maltose-binding protein. A, purification of the
fusion protein. MBP-PL1 was purified by affinity chromatography on
amylose column from lysates of E. coli transformed with
TB1[pMal-PL1] as described under "Experimental Procedures."
Aliquots taken from different stages of the purification were analyzed
by 12.5% SDS-polyacrylamide gel electrophoresis. Lane
1, molecular mass markers; lane 2,
noninduced bacterial lysate; lane 3,
isopropyl-1-thio- -D-galactopyranoside-induced bacterial
lysate; lane 4, protein after amylose affinity
chromatography. The position of MBP-PL1 is indicated. B,
proteolytic activity of MBP-PL1. The trans-cleavage assay using
in vitro generated [35S]Met-labeled substrate
was used to monitor proteolytic activity of purified MBP-PL1
(lanes 1 and 2) and in
vitro generated, nonlabeled polypeptide containing PL1pro
(pp1a/pp1ab-(1-1315)) and its mutated derivative (lanes
3 and 4). After immunoprecipitation of the
cleavage reaction with IS 1720, proteins were separated by 10-17.5%
gradient SDS-polyacrylamide gel electrophoresis, and labeled
polypeptides were visualized by autoradiography. The positions of
molecular mass markers, the substrate (p102), and cleavage products
(p93 and p9) are indicated. The source of enzyme was as follows:
MBP-PL1 C1054S (lane 1), MBP-PL1 (lane
2), in vitro produced PL1pro C1054S (lane
3), in vitro produced PL1pro (lane 4).
Metal content in MBP-PL1
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Fig. 3.
Effect of Zn2+ on the proteolytic
activity of MBP-PL1. The trans-cleavage assay was used to monitor
proteolytic activity of MBP-PL1 subjected to one or two cycles of
denaturation in the presence of 8 M urea and renaturation
in the presence of EDTA, ZnOAc, or CoOAc. The substrate and cleavage
products are indicated as in Fig. 2. Lane 1,
molecular markers. The source of enzyme was as follows: buffer A
(lane 2), MBP-PL1 (not treated) (lane 3),
denatured MBP-PL1 renatured in the presence of EDTA (apoenzyme)
(lane 4), denatured MBP-PL1 renatured in the presence of
ZnOAc (lane 5), denatured apoenzyme MBP-PL1 renatured in the
presence of EDTA (lane 6), denatured apoenzyme
MBP-PL1 renatured in the presence of ZnOAc (lane 7),
denatured apoenzyme MBP-PL1 renatured in the presence of CoOAc
(lane 8).
Proteolytic activity of HCoV PL1pro mutants
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet topology, most probably of the zinc ribbon type, and be
incorporated into a papain-like fold. As an extension of this analysis,
a possible tertiary organization of PL1pro is presented in Fig.
4 as a ribbon model that was built by
homology modeling (52) using the alignments shown in Fig. 1. We believe
that this model is useful for rationalizing the results presented here
(see below) and for the development of more precise mutagenesis
analyses. At the same time, we recognize that the model most probably
deviates significantly from the coordinates of the actual structure.
This lack of robustness is due to the fact that none of the current
methods of homology modeling (52) can rigorously predict the structure
of proteins, when modeling is based upon two separate templates of a
marginal similarity, as was the case for PL1pro. Despite these
reservations about the model, the compatibility of the Zn2+
ribbon and the PL fold is supported by two observations: (i) the
distance between the N and C termini of the Zn2+ ribbon
fits between the two "acceptor" residues of the core PL domains in
papain and (ii) the Zn2+ ribbon spatially replaces the
C-terminal
B-strand in papain, and, accordingly, a counterpart of
this strand is not conserved in the coronaviral PLpros (Fig.
1A). The interdomain junction role of the Zn2+
finger is also compatible with the observation that many RNA viral
PLpros, although not all (26, 74), have considerably smaller sizes than
coronaviral PLpros and may contain only a short interdomain loop in
place of the Zn2+
finger.2 A unique
-sheet
topology of the interdomain junction has most recently been also
described for the FMDV Lpro, the only RNA viral PLpro whose
three-dimensional structure was solved (29).
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Fig. 4.
A crude structural model of HCoV PL1pro.
The PL1pro model (right; pp1a/1ab residues 1033-1242) was
generated using the structures of papain (1ppn; shown left)
and human TFIIS (1tfi) as templates for building the core and a loop
library for constructing the variable regions by using Quanta 97 and
Whatif 4.99. The model was partly refined but not minimized. Secondary
structure was calculated according to Ref. 57 and, for some positions
in PL1pro, from the predicted secondary structure. The structures of
both proteases are displayed (56) in the standard papain orientation
(66) and split into three domains. These are colored
according to a scheme given in Fig. 1C as follows.
Blue, left-hand -helix domain; green,
right-hand
-sheet domain without counterparts of
A- and
B-strands; red, Zn2+ finger domain in PL1pro
and the interdomain loop along with
A- and
B-strands and
-RII-helix in papain. Cysteine residues of the Zn2+
finger as well as the catalytic dyad residues of PL1pro that have been
probed by site-directed mutagenesis (Table II) are shown in the
ball-and-stick model.
The Zn2+ finger occupies one of the most pronouncedly
diverged regions of coronaviral PLpros and seems to form a separate
domain (Figs. 1, A and C, and Fig. 4).
Surprisingly, the proteolytic activity of HCoV PL1pro was abolished by
any of three Zn2+ finger mutations that mimicked the
wild-type sequences of HCoV PL1pro relatives (V1126/27ins, L1155del,
and C1157H in Table II). To reconcile these findings, one must propose
that, in the evolution of the HCoV PL1pro homologues, such mutations
have been suppressed by other replacements in the Zn2+
finger or the catalytic domains. If the changes were limited to the
Zn2+ finger, this would imply that the Zn2+
finger is either involved in substrate binding or controls movement of
the catalytic domains. Both options seem possible given the interdomain
position of the Zn2+ finger and its likely proximity to the
active site (Fig. 4). If the catalytic domains accepted mutations as
well, this would be indicative of an interaction between these domains
and the Zn2+ finger. Again, the current model does not
preclude an interaction between the Zn2+ finger and -LI
helix, which is capped by the catalytic Cys1054 (Fig. 4).
It is conceivable that the PL1pro Zn2+ finger interacts
with both the catalytic domains and substrate.
HCoV PL1pro and, by implication, the other coronaviral PLpros extend the list of nonmetal proteinases dependent on Zn2+ and encoded by RNA viruses. Recently, a Zn2+ has been identified as a structural component in the picornavirus 2A and hepatitis C virus NS3 proteases (17-22). In these enzymes, Zn2+ is uniquely coordinated by residues that have been naturally engineered in two loops of the CLpro fold. In both the chymotrypsin-like or papain-like Zn2+-binding viral proteases, Zn2+ is located at the site opposite to the active center. The recurrent emergence of Zn2+ dependence in the two independent lineages of RNA viral nonmetal proteinases could be linked to their biology (12). For example, in the coronaviral PLpros, the prominent position of the Zn2+ finger makes it an excellent candidate for mediating external signals that might modulate proteolytic activity inside cells. Through the Zn2+ finger, the PLpros might interact with proteins or polynucleotides and, in turn, affect processes regulated by the PLpro partner (75).
Among the positive sense RNA viruses, coronaviruses have the largest
genomes (close to 30 kilobases) and probably use the most sophisticated
mechanisms of RNA replication and transcription (76). As is the case
for poliovirus (7), coronaviral PLpros might turn out to be important
regulators at these levels. It may not be entirely coincidental that
the Zn2+ finger used as the template in our HCoV PL1pro
model employs a variant of the general architecture motif of RNA
polymerases (70). The lack of an infectious DNA clone for any
coronavirus remains a major technical obstacle in our attempts to
elucidate the possible roles of PLpros in the viral replicative cycle.
However, regardless of the role(s) played by PLpros, the integrity of
its unusual domain organization could be targeted by specific drugs for
the control of coronaviral infections.
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ACKNOWLEDGEMENTS |
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We thank B. Schelle for excellent technical assistance, P. Schramel for ICP-OES analyses, M. Gerstein and R. Altman for help with the CORE package, K. Miaskiewicz for the administration of computer resources, and S. Burt and J. Maizel for commenting on the manuscript.
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FOOTNOTES |
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* This project has been funded in part by Deutsche Forschungsgemeinschaft Grant SFB 165/B1 (to S. G. S.), Russian Fund for Basic Research Grant 96-04-49562, a fellowship from the Netherlands Organization for Scientific Research (to A. E. G.), and federal funds from NCI, National Institutes of Health, under Contract NO1-CO-56000.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Microbiology and Immunology, Box 0414, University of California, San Francisco, CA 94143-0414.
To whom correspondence should be addressed: SAIC/NCI-FCRDC, 430 Miller Dr., Rm. 235, Frederick, MD 21702-1201. Tel.: 301-846-1991; Fax:
1-301-846-5762; E-mail: gorbalen{at}ncifcrf.gov.
2 A. E. Gorbalenya, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CLpro, chymotrypsin-like protease; PLpro, papain-like proteinase; aa, amino acid(s); ICP-OES, inductively coupled plasma optical emission spectrometry; MBP, maltose-binding protein; FMDV, foot-and-mouth disease virus; HCoV, human coronavirus 229E; MHV, murine hepatitis virus; TGEV, porcine transmissible gastroenteritis virus; IBV, avian infectious bronchitis coronavirus; CoOAc, cobalt acetate; ZnOAc, zinc acetate.
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