Distant structural homology leads to the functional characterisation of an archeal PIN domain as a 5

35 Pages·2004·0.67 MB·English

JBC Papers in Press. Published on January 20, 2004 as Manuscript M313833200
Distant structural homology leads to the functional characterisation
of an archaeal PIN-domain as an exonuclease.

Vickery L. Arcus1,2,*, Kristina Bäckbro1,4, Annette Roos1,4, Emma L. Daniel1,3 and
Edward N. Baker1,3.

1School of Biological Sciences, 2AgResearch Structural Biology Laboratory and 3Centre
of Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland, New D
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4Present address: Department of Cell and Molecular Biology, Uppsala University, ://w
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Biomedical Centre, Box 596, SE-751 24 Uppsala, Sweden. .o
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*Corresponding author J
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Name: Vickery Arcus 1
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Phone: [phone]
Fax: [phone]
Email: [email protected]

Running title: Structure and function of an archaeal PIN domain

Key words: Structural genomics, PIN-domain, X-ray crystallography, exonuclease.

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
SUMMARY

Genome sequencing projects have focused attention on the problem of discovering the
functions of protein domains that are widely distributed throughout living species but
which are, as yet, largely uncharacterised. One such example is the PIN domain, found in
eukaryotes, bacteria and archaea, and with suggested roles in signalling, RNase editing
and/or nucleotide binding. The first reported crystal structure of a PIN domain (open
reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has
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been determined to 2.5 Å resolution and is presented here. Mapping conserved residues nlo
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from a multiple sequence alignment onto the structure identifies a putative active site. fro
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The discovery of distant structural homology with several exonucleases, including T4 ://w
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phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN .jb
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domains as Mg2+-dependent exonucleases, a hypothesis which we have confirmed in by/
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vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests t o
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a mechanism for selective cleavage of single stranded overhangs or flap structures. These ry
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results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are 1
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strikingly numerous in thermophiles, and in organisms such as Mycobacterium
tuberculosis. They also support previous hypotheses that eukaryotic PIN domains
participate in RNAi and nonsense mediated RNA degradation (NMD).

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INTRODUCTION

The explosive growth of whole genome sequencing efforts, and the discovery that a large
proportion of the assumed gene products are of unknown or poorly-understood function,
has focused attention on new approaches to assigning function. In the absence of
sufficient sequence similarity to clearly infer homology with already characterised
proteins, a variety of bioinformatic approaches have been used to obtain functional clues.
These include, for example, analyses of genome location (seeking potential operons),
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phylogenetic profiling, and observations of gene fusions in different species (1,2). An wn
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alternative, complementary, approach is to use analyses of protein three-dimensional fro
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structure to derive functional insights, since three-dimensional structure is conserved in ttp
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evolution much more strongly than sequence. This provides a rationale for a number of .jb
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structural genomics initiatives (3-6). b/
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As part of a pilot structural genomics project aimed at the discovery of biological a
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function, we have focused on gene products from the hyperthermophilic crenarchaeon , 20
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Pyrobaculum aerophilum, an organism whose complete genome sequence was published
recently (7). A whole-genome comparison of P. aerophilum and Mycobacterium
tuberculosis, two organisms with very different, and in a sense extreme, lifestyles, led us
to identify a set of 250 pairs of orthologous genes that are both widely distributed in
nature and are shared by these two organisms. Among these were a set of four genes from
P. aerophilum (PAE0151, PAE0285, PAE0337, PAE2754) and four from M. tuberculosis
(Rv0065, Rv0549, Rv0960, Rv1720) that have since been clustered at NCBI as part of
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COG4113, with members drawn from archaea, cyanobacteria, actinobacteria and alpha
proteobacteria (Table 1). These are now annotated in Pfam as PIN domains.
[Table 1]
PIN domains, named for their homology with the N-terminal domain of the pili
biogenesis protein (PIN = PilT N-terminus) (8), comprise a very large family of proteins
with representatives in all three kingdoms of life. They are classified in the Pfam database
(http://www.sanger.ac.uk/Software/Pfam) as PF01850, currently with more than 340
members. Functional annotation of the PIN domains is equivocal. They were initially
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thought to function in signalling (9), but a recent bioinformatic analysis of nearly 100 ow
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PIN domain sequences identified a set of five conserved acidic residues and a sixth ed
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conserved position at which there is either a Serine or Threonine residue. This has led to a http
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suggested exonuclease function (10). In eukaryotes, for example, the C. elegans PIN w
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domain, smg-5, and the yeast PIN domain, NMD4p, are postulated to be ribonucleases brg/
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(RNases) that bind to helicases as part of the machinery for RNA degradation via the es
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RNAi and nonsense mediated RNA degradation (NMD) pathways (10). nu
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In archaea and thermophilic bacteria, PIN domains have been associated with a possible
role in DNA repair. A recent analysis of conserved gene context across fully sequenced
prokaryotic genomes revealed, in most archaea and some thermophilic bacteria, a
previously unrecognised cluster of genes containing DNA polymerases, helicases,
nucleases and many conserved hypothetical open reading frames, one of which is a PIN
domain clustering in COG1848 (11). This suggested a new DNA repair system in these
organisms. DNA repair, and particularly mismatch repair, in thermophiles is a vexing
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question. The absence of key mismatch repair enzymes such as MutS and MutL, which
are highly conserved in mesophiles from E. coli to humans, has been suggested to result
in “mutator” lifestyles for some thermophiles (7), in which adaptive mutations enable the
organism to adapt to stress or extreme environments (12). Also absent from many of the
fully sequenced thermophilic genomes are several well conserved nucleotide excision
repair enzymes. The discovery of a new DNA repair operon in archaea addresses this
question and by implicating PIN domains as part of this operon adds another piece of
functional evidence for this large protein family.
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Here we present the first crystal structure of a PIN domain, from the crenarchaeon ed
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Pyrobaculum aerophilum. The protein, the gene product of open reading frame http
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PAE2754, proves to be a distant structural homologue of T4 RNase H and other w
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exonucleases, despite insignificant sequence identity. Strict conservation of the active site brg/
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residues suggests that this PIN domain is indeed an exonuclease. We have confirmed this es
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functional hypothesis in vitro. This has important implications both for archaeal DNA nu
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editing and for the role of PIN domains in eukaryotic RNA editing. It is also an , 2
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illustration of the power of structural genomics whereby deep phylogenetic lineages are
apparent at the structural level and lead directly to functional characterisation of proteins
of previously unknown function or with equivocal and/or general functional annotation.

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EXPERIMENTAL PROCEDURES
Protein Expression, Purification and Crystallization
The predicted open reading frame PAE2754 was amplified from genomic DNA by PCR,
subcloned into the expression vector pPROeX (Lifetech), transformed into E. coli
BL21(DE3) cells and expressed as an N-terminal His -tagged protein. Purification
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involved a heat step, in which incubation for 40 minutes at 80 ºC denatured a large
fraction of the E. coli proteins, followed by Ni2+ affinity chromatography and size
exclusion chromatography, as described (13).
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Two site-specific mutations, L65M and L80M, were designed and introduced to facilitate from
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structure determination by multiwavelength anomalous diffraction (MAD) methods using ://w
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the selenomethionine (SeMet)-substituted protein. The single mutants were individually c
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made and tested for expression and crystallization, followed by the double mutant y g
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L65M/L80M. Mutagenesis was performed with the QuikChange site-directed n J
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mutagenesis kit (Stratagene). The double mutant was stable at 80 ºC suggesting that the ry 1
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structure was not significantly destabilised by these mutations. The plasmid encoding the 9
double mutant was transformed into the methionine auxotroph E. coli strain DL41(DE3)
and grown in LeMaster medium with SeMet as the only methionine source. The SeMet-
substituted double mutant protein (SeMet-PAE2754_MM) was then purified as above.

Both native PAE2754 and SeMet-PAE2754_MM were crystallized as described (13) and
flash-cooled for data collection by soaking in cryoprotectant (mother liquor plus 10%
glycerol) immediately prior to placement in a stream of cold N gas at 110 K.
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Structure Determination and Refinement
Native PAE2754 X-ray diffraction data to 2.5 Å resolution were collected at the National
Synchrotron Light Source (NSLS), Brookhaven, on beamline X8C (λ=1.0000 Å). MAD
data at two wavelengths were collected for SeMet-PAE2754_MM at the Stanford
Synchrotron Radiation Laboratory (SSRL), beamline 9-1. The data were processed using
DENZO and SCALEPACK (14). Data collection and refinement statistics are given in
Table 2. The structure of PAE2754_MM was determined by single anomalous diffraction
(SAD) using a single SeMet dataset at λ=0.9794 Å. This is not where the anomalous
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differences are maximised for Se, but was necessary because the “remote” wavelength n
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data set proved to be of poor quality due to crystal decay (data were initially collected at fro
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two wavelengths in accordance with (15)). Using SOLVE (16,17), a total of 17 out of a ttp://w
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possible 24 Se sites (from the 8 monomers in the crystal asymmetric unit) were located, .jb
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based on anomalous differences. These gave initial phases to 2.8 Å with a figure of merit by/
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of 0.18 and a Z-score of 20.6. The phases were improved using maximum likelihood t o
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density modification via RESOLVE (16). Successful improvement of the phases was ary
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dependent on user-defined non-crystallographic symmetry elements based on the initial 01
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Se positions.

Much of the core structure (approximately 74 residues per monomer) was built
automatically with RESOLVE (16) and TEXTAL (18,19), and the rest of the structure
was built manually, during twelve cycles of model building with O (20) and refinement
with CNS (21). Due to the relatively low resolution of the data, non-crystallographic
symmetry (NCS) was included as a restraint in all refinement cycles and weighted
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experimental phases from RESOLVE were also included at all stages. The final
PAE2754_MM structure was then used as a molecular replacement model for the native
data and this structure was completed using 5 cycles of model building and refinement.
Refinement statistics for both models are given in Table 2.
[Table 2]
In vitro exonuclease assays
An 18 base pair (bp) primer (5'-CGCGCCGTTGCTATCTCC-3') was annealed to a 54 bp
primer (5'-ATTGAGAAATTCACGGCGNNKATANNKNNKGTTNNKGGAGA
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TAGCAACGGCGCG-3'; N=T,G,A,C; K=T,G) to form double stranded DNA with a wn
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36 bp, 5'-3' single stranded, randomised overhang. 200 pM of DNA was then mixed with fro
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200 pM of PAE2754 in 20 mM NaCl and 10 mM MgCl (MgCl was omitted from the ttp
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negative control) and incubated at 37 ºC. The reaction was stopped at different time .jb
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points by the addition of formamide gel loading buffer (80% v/v formamide, 10 mM b/
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EDTA), followed by freezing at -20 ºC. Samples were run on a 20% polyacrylamide-urea t o
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denaturing minigel and visualised using ethidium bromide staining. Samples were also a
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prepared in the same way using MnCl as the metal ion source. , 20
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RESULTS
Crystal structure of PAE2754
The crystal structure of the protein encoded by the open reading frame PAE2754 from
P. aerophilum was solved at 2.8 Å resolution, as a selenomethionine (SeMet)-labelled
double mutant, incorporating two L→M mutations, that was constructed to facilitate
phasing by MAD methods. This derivative structure was then refined to give a final R-
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factor of 0.226 (R = 0.279). The native structure was then solved by molecular
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replacement and refined at 2.5 Å resolution to a final R-factor of 0.250 (R = 0.305).
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The resulting model has good stereochemistry (Table 2) with 92% of residues in the most
favoured region of the Ramachandran plot.

The PAE2754 monomer forms a single domain in which the 133-residue polypeptide is
folded as an α/β/α stack, with a central twisted parallel β-sheet of five short strands
(Figure 1). The strand order is 32145 and the twist of the sheet is such that the outer
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strands, which involve just three residues each, are oriented at 160º with respect to each w
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other. Between strand 2 and strand 3, helices α2 and α3 pack in an antiparallel manner to d fro
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form a long protrusion that extends orthogonally from the α/β/α stack. Hydrophobic ttp
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cores above and below the central β-sheet stabilise the α/β/α stack and a third .jb
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hydrophobic mini-core is formed below the stack by the orthogonal packing of helices α4 by/
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and α5. These hydrophobic cores are highly populated by Ala, Leu and Val residues, t on
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which comprise no less than 40% of the PAE2754 sequence. ry
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Dynamic light scattering measurements show that PAE2754 forms a tetramer in solution,
and accordingly the 12 monomers in the crystal asymmetric unit are organised as three
tetramers (8 monomers in the asymmetric unit are organised as two tetramers for
PAE2754_MM). The tetramer is best described as a dimer of dimers (Figure 2). An
extensive dimer interface is formed by the two-fold related packing of a nearly
continuous region of sequence between residues 32 and 94, spanning helices α2, α3 and
α4. This interface buries 1440 Å2 of surface area (19% of the total monomer surface) and
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is dominated by hydrophobic interactions, marked by a striking interdigitation of many
large hydrophobic sidechains. There are just six hydrogen bonds between the two protein
chains, all centred around the stacked histidine aromatic rings at the centre of the
interface.
[Figure 2]
The tetramer is formed by the association of two dimers via a relatively small interface,
in which the C-terminus of helix α2 from one monomer contacts the N-terminus of helix
α6 from another. Stabilising interactions at this interface are modest, and include a salt
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bridge between Arg48 from one monomer and Asp110 and Glu112 from another. The w
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backbone carbonyl group from Arg48 also hydrogen bonds across the interface to the d fro
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amide group of Arg111. Although only 450 Å2 of surface area per monomer is buried on ttp
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formation of this interface, the cooperative association of two dimers buries in total .jb
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4x450=1800 Å2. There is also a diagonally stabilising interaction across the tetramer brg/
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whereby a chloride ion linearly coordinates two arginine residues. The chloride lies at st o
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the centre of a sphere of charged side-chains which include Arg48 (chain A), Glu112 and ua
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Lys116 (chain B), Glu112 and Lys116 (chain C), and Arg48 (chain D). , 20
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Residues that are conserved across COG4113 (Figures 2 and 3) are clustered in a pocket
formed at the C-terminal end of the β-sheet and the N-termini of helices α2 and α6. This
arrangement brings together four conserved acidic residues (Asp8, Glu38, Asp92 and
Asp110 in PAE2754) that point into the pocket and create a highly negatively charged
hole. Two other conserved residues on either side of Asp110, Thr108 and Leu112, also
flank the acidic pocket with Thr108 being hydrogen bonded to Asp8. In the PAE2754
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