A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions

Article abstract of DOI:10.1021/acsinfecdis.6b00079

Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.

Full text of DOI:10.1021/acsinfecdis.6b00079

Letter
pubs.acs.org/journal/aidcbc
A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus
DNA Polymerase Subunit Interactions
Han Chen,† Molly Coseno,†,Δ Scott B. Ficarro,‡ My Sam Mansueto,†,§ Gloria Komazin-Meredith,†,#
Sandrine Boissel,†,⊥ David J. Filman,† Jarrod A. Marto,†,‡ James M. Hogle,† and Donald M. Coen*,†
†Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston,
Massachusetts 02115, United States
‡Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston,
Massachusetts 02215, United States
S
* Supporting Information
ABSTRACT: Human cytomegalovirus DNA polymerase
comprises a catalytic subunit, UL54, and an accessory subunit,
UL44, the interaction of which may serve as a target for the
development of new antiviral drugs. Using a high-throughput
screen, we identified a small molecule, (5-((dimethylamino)-
methylene-3-(methylthio)-6,7-dihydrobenzo[c]thiophen-
4(5H)-one), that selectively inhibits the interaction of UL44
with a UL54-derived peptide in a time-dependent manner, full-
length UL54, and UL44-dependent long-chain DNA synthesis.
A crystal structure of the compound bound to UL44 revealed a
covalent reaction with lysine residue 60 and additional
noncovalent interactions that cause steric conflicts that
would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model
substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the
compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may
serve as a starting point for new, needed drugs to treat human cytomegalovirus infections.
KEYWORDS: human cytomegalovirus, DNA polymerase, protein−protein interaction, covalent inhibitor, SGM8
rotein−protein interactions (PPIs) are central to all
and UL44 are essential for HCMV DNA replication, and their
interaction is well-defined.12−18 The crystal structure of UL44
bound to a peptide corresponding to the C-terminal 22 amino
acids of UL54 (P54) reveals intersubunit contacts including a
crevice on UL44, into which hydrophobic residues of UL54
insert, and the UL44 “connector loop” (defined below), with
which UL54 extensively hydrogen bonds.15 Importantly, single
substitutions of residues on the interaction interface can
dramatically impair the PPI and long-chain DNA synthesis,16,19
supporting this PPI as a potential drug target. Inhibitors of this
PPI should be active against viruses resistant to current anti-
HCMV drugs.
Previously, a screen of ∼50,000 compounds in five libraries
identified small molecules that specifically disrupt the UL54−
UL44 PPI in vitro and inhibit HCMV replication in cell
culture.9 However, the compounds identified had less than ideal
properties for further development, with structures that include
three or more fused aromatic rings (the structure provided by
the supplier of one of these compounds (AL21) did not have
three such rings, but further analysis showed that the
P
biological processes and present a vast class of potential
therapeutic targets for pharmacological intervention. Although
targeting PPIs with small molecules can be challenging,
considerable progress has been made in identifying PPIs that
are amenable to such inhibition.1,2
We have been studying the feasibility of pharmacologically
disrupting PPIs of human cytomegalovirus (HCMV). HCMV
rarely causes disease in immunocompetent individuals, but in
̈
neonates, who are immune-naıve, and immunocompromised
patients, HCMV can cause serious diseases.3 Although
currently approved drugs can successfully treat certain
HCMV diseases, limited efficacy, toxicity, and the emergence
of drug resistance limit their use.4 Thus, better anti-HCMV
agents active against mutants resistant to current drugs are
needed.
An important HCMV PPI is that between the viral DNA
polymerase catalytic subunit, UL54,5 and an accessory subunit,
UL44, which stimulates long-chain DNA synthesis, presumably
by increasing processivity.6,7 There has been considerable
interest in compounds that inhibit PPIs between polymerase
catalytic subunits and processivity factors (e.g., refs 8−11),
highlighted by the recent discovery that griselimycin inhibits
Mycobacterium tuberculosis by this mechanism.10 Both UL54
Received: May 14, 2016
Published: November 25, 2016
© XXXX American Chemical Society
A
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Figure 1. (a) Chemical structure of SGM8. (b) Activity of SGM8 against the interactions of UL44−P54, UL42−P30, and PCNA−P21 in FP assays.
The normalized mP at each concentration of SGM8 relative to the control without inhibitor was plotted versus the concentration of SGM8. FP was
assayed following pre-incubation for the times indicated in each panel. (c) (Top) SGM8 inhibition of binding of UL44 to full-length UL54, as
analyzed by biolayer interferometry; (bottom) SGM8 inhibition of long-chain DNA synthesis mediated by UL54 in the presence of UL44 (UL54/
UL44, squares; see Figure S2) or DNA synthesis by UL54 alone in a filter-binding assay (circles), relative to the signal in the absence of compound.
Error bars show standard errors of the mean.
compound did). Medicinal chemistry efforts to improve the
properties of these compounds did not succeed.
thio)-6,7-dihydrobenzo[c]thiophen-4(5H)-one), that we called
SGM8 (Figure 1a; structure, purity, and stability verified by
NMR, HRMS, and LC-MS (Figure S1 and Supporting
Information (SI) text). Despite its expected reactivity, we
were intrigued by its apparent selectivity and relatively small
size.
While performing dose−response studies, we found that
when we incubated samples at room temperature for 10 min
prior to measurement, SGM8 only weakly inhibited the P54−
UL44 interaction, with an IC50 ∼ 20 μM, similar to that for the
P30−UL42 interaction (Figure 1b, top and middle). However,
when we extended the incubation time to 20 min or beyond,
SGM8 inhibited the P54−UL44 interaction with an IC50 of 2
μM (Figure 1b, top). Extending the incubation time to 60 min
only slightly increased SGM8 inhibition of the P30−UL42
interaction (Figure 1b, middle) and did not meaningfully
change the very weak inhibition by SGM8 of the P21−PCNA
interaction (Figure 1b, bottom). Thus, SGM8 selectively
inhibits the P54−UL44 interaction in a time-dependent
manner.
To try to identify starting points for more “drug-like”
compounds that target the UL54−UL44 PPI, we screened 45
other libraries with a total of 213,457 small molecules or
extracts at the Harvard Institute of Chemistry and Cell
BiologyLongwood (ICCB-L), utilizing our previously
reported fluorescence polarization (FP) assay.8,9 The initial
screen assayed the interaction of fluorescently labeled P54 and
glutathione S-transferase (GST) fused UL44ΔC290 (herein
referred to as UL44). Hits from this initial screen (414
compounds) were rescreened and subjected to two counter-
screens to assess selectivity. As UL44 displays a fold strikingly
similar to other processivity factors,20 such as UL42, the herpes
simplex virus-1 (HSV-1) processivity subunit,21 and proliferat-
ing cell nuclear antigen (PCNA), the eukaryotic processivity
factor,22 we tested the hits for inhibition of the interaction of
HSV-1 UL42 and fluorescently labeled P30, a peptide
corresponding to 36 residues of HSV-1 polymerase UL30,23
and the interaction of human PCNA and fluorescently labeled
P21, a 22 residue peptide derived from the cell-cycle checkpoint
protein p21,22 in FP assays similar to the UL44−P54 assay.
These assays, which entailed prolonged incubation of the
components prior to assay, identified 23 compounds including
a small molecule, (5-((dimethylamino)methylene-3-(methyl-
We also found that SGM8 inhibits the interaction of UL44
with full-length HCMV GST-UL54 (herein referred to as
UL54) with an IC50 of 4.5 μM in a biolayer interferometry
assay (Figure 1c, top). We then tested whether SGM8 inhibits
long-chain DNA synthesis by UL54−UL44,16,19 using a
B
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poly(dA)-oligo(dT) primer-template. As previously described,9
we observed only short-chain DNA synthesis with UL54 alone
(Figure S2, lane 1), but formation of long DNA products with
UL54 and UL44 (Figure S2, lane 2) that could be inhibited by
P54 (Figure S2, lanes 3 and 4). SGM8 inhibited long-chain
DNA synthesis in a dose-dependent manner (Figure S2, lanes
5−10; Figure 1c, bottom), with an IC50 of 3.2 μM, similar to
that observed in the FP and biolayer interferometry assays.
However, SGM8 exhibited much less potent inhibition (IC50
>
100 μM; Figure 1c, bottom) of DNA synthesis by UL54 alone,
using the same primer-template in a filter binding assay.18 Thus,
SGM8 inhibition was selective for long-chain DNA synthesis
mediated by UL44.
We prepared crystals of UL44 at neutral pH in the C2221
space group (the space group of the crystals used in the
structure of the UL44−P54 complex15), soaked them with
SGM8, and determined the crystal structure of the complex
(PDB code: 5IWD) to 2.56 Å resolution (Rwork/Rfree, 0.2125/
0.2670) by molecular replacement (Table S1), initially based
on the low-pH crystal form of the UL44−P54 complex
structure.15
UL44 is a head-to-head dimer.20 Each monomer contains
two topologically similar domains (N-terminal and C-terminal)
linked by a connector loop (Figure S3A), with a “front” face
(including the connector loop) that interacts with UL54.20 The
structure of the complex revealed a strong covalent SGM8
binding site that includes residue K60 on the N-terminal
domain below the connector loop and two weaker sites at each
end of the monomer, one that includes K192 and one that
includes K4 (Figure S3A). Interestingly, there are 12 other
lysines visible in the structure, of which at least 10 are solvent-
exposed and completely accessible. Five of these 10 lie close to
hydrophobic residues. The three SGM8 substitution sites are
each close to hydrophobic crevices. The weaker sites share a
hydrophobic crevice created by crystal packing contacts and the
hydrophobic portions of the two SGM8 molecules; thus, this
hydrophobic crevice would not be expected to form in solution.
Density corresponding to SGM8 at the strong site was readily
visualized (Figure S3B). Notably, the density corresponding to
SGM8 no longer fit the original compound, but instead fit a
rearranged form of SGM8 in which the dimethylamino group is
lost, and the compound forms a covalent adduct on the side
chain of K60 (Figure S3B). The SGM8 adduct extends from
K60 into a hydrophobic slot between the connector loop and a
β sheet, making numerous noncovalent interactions (Figure 2a,
stereoview in Figure S3C). Interestingly, there are no contacts
between SGM8 and residues in or near the cleft of UL44 (e.g.,
V136 and F266 (Figure 2a and Figure S3C)) into which certain
hydrophobic residues of UL54 insert.15 Instead, the secondary
amino group of the covalent adduct hydrogen bonds with
residue Q51, while the 3-(methylthio)-6,7-dihydrobenzo[c]-
thiophen-4-one moiety makes numerous hydrophobic contacts
with UL44 residues on both sides of the slot, including contacts
with the main chain and side chain of I135 of the connector
loop and weak but extensive contacts with the side chains of
T41, L43, I49, and T79 on the β-sheet (Figure 2a and Figure
S3C). These noncovalent contacts likely contribute to binding
affinity and help position SGM8 for reaction with the K60 side
chain.
Figure 2. How SGM8 interacts with UL44 and interferes with P54
binding. (a) SGM8 covalently bonds with UL44 residue K60 (orange)
and noncovalently interacts with UL44 in a hydrophobic slot, making
contacts with residues 133−137 of the connector loop (cyan) and with
side chains from the central β sheet (yellow and green). In this ribbon
representation, important contacts are shown as stick models and are
labeled. The Ile135 side chain extends toward the viewer. (Stereoview
in Figure S3C.) (b) Binding of P54 (dark purple) to UL44 (PDB
entry: 1YYP),15 includes β-sheet hydrogen bonding with the
connector loop (cyan and pink). Residues 133−137 (cyan) are
oriented differently from panel a, with the Ile135 side chain extending
laterally. (c) In this modeling exercise, the peptide-binding connector
loop (from 1YYP) replaces residues 128−143 in the SGM8−UL44
complex. A steric clash between SGM8 and Ile135 prevents the
connector loop from adopting its peptide-bound conformation.
The contacts of SGM8 with I135 were particularly interesting
because substitution of that residue with alanine (I135A)
ablates interaction with HCMV UL54.19 In the structure of
UL44 bound to P54 (Figure 2b), I135 makes extensive
interactions with the β-sheet below the connector loop,
particularly with residues that interact with SGM8 in the
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Figure 3. Reactivity of SGM8 with model substrates. (a) (Left) LC-MS analysis of the reaction of SGM8 with pentylamine following incubation for 0
min, 15 min, and 24 h; (right) proposed reaction mechanism for the reaction converting SGM8 to the species eluting at 10.2 min (SGM8A) and
below it the proposed reaction mechanism for the reaction between SGM8 and pentylamine to produce the species eluting at 11.2 min (SGM8-
pentylamine). (b) MALDI-TOF analysis of the reaction of peptide P-1 with SGM8. (Top) Peaks at 1047 and 1255 Da (∗) were assigned to
unreacted P-1 and SGM8-conjugated peptide P-2 (upper panel). Proposed reaction is shown in the inset. The peak labeled Met Ox corresponds to
unreacted P-1 with oxidized methionine. (Bottom) MALDI-MS/MS spectrum of labeled peptide P-2 (lower panel). Ions of types b and y are shown
in blue and red, respectively. The difference between y5 and y6 is 336 Da, corresponding to the mass of a lysine residue conjugated to SGM8 with
loss of the dimethylamino group.
present structure, to anchor the connector loop so that it can
form a hydrogen-bonding network between the connector loop
and the Pol peptide P54.15 That connector loop conformation
differs substantially from that when SGM8 is bound (compare
Figure 2a,b). Indeed, as illustrated in Figure 2c, which
superimposes the position of SGM8 from Figure 2a with the
position of the connector loop bound to P54 in Figure 2b, the
connector loop would clash with the covalently bound
compound. This explains how SGM8 inhibits P54 binding to
UL44.
We also solved the structure of unliganded UL44 at neutral
pH (PDB code: 5IXA) from crystals in space group P212121
(obtained under slightly different conditions) to 2.7 Å
resolution, and, in that structure, the connector loop is not
prohibited from interacting with P54 (Figure S4 and
accompanying SI text).
pH (pH 7.5) used for crystallography. We used LC-MS to
monitor this reaction (Figure 3a). When the reaction contained
only SGM8, we detected a main peak at 10.2 min with MW
227, lower than what we expected for SGM8 (MW 253). This
peak corresponds to a derivative of SGM8 that we term
SGM8A, the identity of which we confirmed using NMR
(Figure S5). SGM8A would be generated by reaction of SGM8
with water under acidic conditions during elution with 0.1%
trifluoroacetic acid in acetonitrile by LC-MS (Figure 3a).
Addition of pentylamine to the reaction generated a new peak
at 11.2 min with MW 296 (Figure 3a), consistent with the MW
expected if SGM8 reacted as an enamine with pentylamine
(structure confirmed by NMR (Figure S6)), generating
dimethylamine as the other product. We then investigated
whether SGM8 can react with a lysine within a peptide, again at
pH 7.5. MALDI-TOF/TOF MS revealed a new species with a
mass 208 Da greater than that of the peptide (Figure 3b, top),
consistent with covalent addition of SGM8 to the peptide, and
loss of the dimethylamino group. MALDI-MS/MS analysis
localized the covalent adduct to the lysine residue in the
To investigate how SGM8 can form covalent bonds with
lysines, we performed reactions using model substrates. In the
first reaction, we incubated SGM8 either by itself or with
pentylamine, which mimics the side chain of lysine, at the same
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peptide (Figure 3b, bottom). These results support a
mechanism in which SGM8 acts as an enamine to form a
covalent bond with K60 on UL44 via a resonance-stabilized
mechanism.
due to reactivity with cellular targets. Development of more
potent and selective inhibitors may permit studies of covalent
modification of UL44 in HCMV-infected cells. Whether or not
advantages of covalent modification in terms of high affinity
and prolonged inhibition could outweigh disadvantages in
terms of time dependence of inhibition and potential off-target
effects including haptenization, given its relatively small size and
the opportunities to use structure-based methods to design
additional contacts (possibly resulting in high affinity non-
covalent inhibitors), SGM8 may provide an interesting starting
point for the discovery of new drugs to treat HCMV infections.
A prediction of this proposed mechanism is that substitution
of K60 with alanine would greatly reduce SGM8 activity. This
substitution had little or no effect on the interaction of UL44
with P54 (Figure S7). However, as predicted, the interaction of
the mutant protein with P54 was substantially less sensitive to
SGM8 than was the wild type UL44−P54 interaction, with
<50% inhibition observed even at 20 μM SGM8 following 60
min of incubation (Figure 4; compare to Figure 1b, top). Thus,
K60 is important for SGM8 inhibition of the interaction, most
likely because SGM8 covalently bonds to this residue.
METHODS
■
FP Assays and High-Throughput Screening. FP assays
of the interaction of UL44 with P5418 and of UL42 with P3023
were used for high-throughput screening as described
previously9 of small molecules of the libraries listed in the SI.
Selected compounds were also tested in a similar FP assay of
the interaction of PCNA with p21 and in dose−response
assays. Details regarding preparation of the proteins and
peptides, and the FP assays are provided in the SI.
UL54−UL44 Interaction Assay. The effect of SGM8 on
the interaction of UL44 and full-length UL54 was assessed
using biolayer interferometry with a BLItz system (forteBIO)
́
following the manufacturer’s instructions, with modifications
and details provided in the SI.
DNA Polymerase Assays. The effects of SGM8 on long-
chain DNA synthesis and basal polymerase activity of UL54 in
the absence of UL44 were performed as described
previously16,18 with the modifications detailed in the SI.
Crystallography. One microliter of 10 mg/mL UL44 in
storage buffer (20 mM Tris (pH 7.5), 500 mM NaCl, 0.1 mM
EDTA, 2 mM DTT, and 5% glycerol) and 1 μL of
crystallization buffer (75 mM ammonium sulfate, 50 mM
HEPES (pH 7.5), and 10% PEG4K) were mixed and
crystallized in hanging drops over 1.8 M NaCl. To introduce
SGM8 into the crystals, crystals were soaked in crystallization
buffer supplemented with 10 mM compound for 3 days at
room temperature. Soaked crystals were flash-frozen in liquid
nitrogen.
Diffraction data were collected at the Argonne National
Laboratory APS ID24C beamline at 100 K. Data were
processed with HKL200027 and merged to yield an essentially
complete data set in space group C2221, extending to 2.56 Å.
Initial molecular replacement phases were based on portions of
the UL44ΔC290 complex with peptide P54 (PDB code:
1YYP),15 which are nearly isomorphous. Model reconstruction
and refinement were performed using Phenix,28 Refmac5,29 and
Coot.30 SGM8, the connector loop, and weaker density features
were added during subsequent refinement cycles. Topology and
parameter files for the inhibitors were generated using
PRODRUG.31 Figures depicting crystal structures were
prepared using PyMol (The Pymol Molecular Graphics System,
Figure 4. Activity of SGM8 against the interaction of mutant UL44
K60A with P54 in FP assays. FP was assayed following the different
pre-incubation times indicated.
In summary, we identified a novel small compound, SGM8,
which, despite its reactivity, selectively inhibits the interaction
of subunits of HCMV DNA polymerase via covalent
modification of K60 of the UL44 subunit. In particular,
SGM8 selectively inhibits the UL44−P54 interaction relative to
other processivity subunit−peptide interactions, selectively
inhibits long-chain DNA synthesis mediated by UL44 relative
to short-chain DNA synthesis by UL54 alone, and selectively
modifies 1 lysine out of at least 13 surface-exposed lysines. That
lysine is normally adjacent to a hydrophobic slot into which
SGM8 can bind with reasonable affinity. As noted, SGM8
binding does not directly block UL54 from interacting with the
UL44 hydrophobic crevice and connector loop. Rather,
compound binding acts allosterically, clashing with the UL44
connector loop to prevent its anchoring, thereby disrupting
hydrogen bonding of the connector loop to UL54. Covalent
bond formation with K60 likely accounts for the time-
dependent inhibition of the interaction, almost certainly
increases binding affinity substantially, and, as there are no
lysines in similar positions in UL42 and PCNA, may impart
selectivity.
To our knowledge, there have been very few examples of
allosteric inhibitors that inhibit PPIs via covalent bond
formation24 and no covalent inhibitors of polymerase subunit
interactions. Aside from the substantial interest in inhibitors of
polymerase subunit interactions as antibacterial and antiviral
drugs,8−11 there is renewed interest in covalent modifiers as
drugs or as starting points for drug design.25 In this regard, it is
interesting that SGM8 targets a lysine rather than a cysteine.26
We screened SGM8 for anti-HCMV activity and cytotoxicity
and found similar potencies in both assays (Table S2), likely
version 1.3, Schrodinger, LLC).
̈
Analyses of SGM8 Reaction with Model Substrates.
SGM8 (2.5 mM) was reacted with excess pentylamine, or 20
nmol of SGM8 was reacted with 2 nmol of the lysine
containing peptide shown in Figure 3. Reaction products were
analyzed by LC-MS and NMR or MALDI MS, respectively, as
detailed in the SI.
Antiviral and Cytotoxicity Assays. Screening for anti-
HCMV activity was conducted using a 3-day assay in HFF cells
that employed a virus (HCMV-pp28-LUC) which expresses
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luciferase under the control of a promoter that requires viral
DNA synthesis for activity. Details regarding construction of
the virus and the assay are provided in the SI. Cytotoxicity was
assessed in HFF cells following a 3-day incubation using a
WST-1 assay (Roche), according to the manufacturer’s
instructions. Details of the assay are provided in the SI.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
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(crystallographic data and screened libraries, respec-
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Figures S1−S7 (NMR spectra (S1, S5, S6), LC-MS data
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(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*(D.M.C.) E-mail: don_coen@hms.harvard.edu.
ORCID
Donald M. Coen: 0000-0002-2148-5671
Present Addresses
(10) Kling, A., Lukat, P., Almeida, D. V., Bauer, A., Fontaine, E.,
Δ(M.C.) University of Vermont, Burlington, VT 05405, USA.
§(M.S.) Merck, Boston, MA 02115, USA.
#(G.K.-M.) Pennsylvania State University, University Park, PA
16802, USA.
Sordello, S., Zaburannyi, N., Herrmann, J., Wenzel, S. C., Konig, C.,
̈
Ammerman, N. C., Barrio, M. B., Borchers, K., Bordon-Pallier, F.,
Bronstrup, M., Courtemanche, G., Gerkits, M., Geslin, M., Hammann,
̈
P., Hernz, D. W., Hoffmann, H., Kiieber, S., Kohlmann, M., Kurz, M.,
Lair, C., Matter, H., Nuermberger, E., Tyagi, S., Fraisse, L., Grosset, J.
⊥(S.B.) Greenlight Biosciences, Medford, MA 02155, USA.
H., Lagrange, S., and Muller, R. (2015) Targeting DnaN for
̈
Author Contributions
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S.B. acquired data; all authors interpreted data; H.C., D.J.F.,
and D.M.C. drafted the manuscript; and all authors critically
revised the manuscript.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Walt Massefski of the Dana-Farber Cancer Institute
NMR core, Tom Wyche of the Harvard Medical School
analytical chemistry core, and Jinhua Wang of Nathanael Gray’s
laboratory for help in analysis of SGM8; Michael J. Eck for
kindly providing a robotic system for high-throughput screen-
ing of initial crystallization conditions; Cai-Hong Yun for
assistance in crystallization and refinement; Nathanael Gray for
advice, assistance, and a critical reading of the manuscript; and
James Marvel-Coen for editorial help. We are especially grateful
to personnel at ICCB-L for assistance with high-throughput
screening, Robin Ross of the Biomolecule Production Core of
NERCE (U54 AI057158) for production of UL44 and UL42,
and Kelly Arnett at the Harvard Medical School Center for
Molecular Interactions for help with BLItz. This work was
supported by NIH Grant AI019838 to D.M.C. and J.M.H., a
sponsored research agreement from Biota Holdings Limited to
D.M.C., and the Dana-Farber Strategic Resources Initiative to
J.A.M.
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