protein structure communications

Acta Crystallographica Section F

Structural Biology
Structure of nicotinic acid mononucleotide
and Crystallization adenylyltransferase from Bacillus anthracis
Communications
ISSN 1744-3091

Shanyun Lu,a Craig D. Smith,a,b Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is
Zhengrong Yang,a Pamela S. the penultimate enzyme in the biosynthesis of NAD+ and catalyzes the
Pruett,a Lisa Nagy,a Deborah adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic
McCombs,a Lawrence J. acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable
DeLucas,a,c Wayne J. candidate for antibacterial drug development; as such, Bacillus anthracis
Brouillettea,d and Christie G. NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for
the purpose of inhibitor discovery and crystallography. The crystal structure of
Brouillettea,d*
BA NaMNAT was determined by molecular replacement, revealing two dimers
a
per asymmetric unit, and was refined to an R factor and Rfree of 0.228 and 0.263,
Center for Biophysical Sciences and
respectively, at 2.3 Å resolution. The structure is very similar to that of B. subtilis
Engineering, University of Alabama at
Birmingham, Birmingham, NaMNAT (BS NaMNAT), which is also a dimer, and another independently
Alabama 35294-4400, USA, solved structure of BA NaMNATrecently released from the PDB along with two
b
Department of Vision Sciences, University of ligated forms. Comparison of these and other less related bacterial NaMNAT
Alabama at Birmingham, Birmingham, structures support the presence of considerable conformational heterogeneity
Alabama 35294-4400, USA,
c and flexibility in three loops surrounding the substrate-binding area.
Department of Optometry, University of
Alabama at Birmingham, Birmingham,
Alabama 35294-4400, USA, and
d
Department of Chemistry, University of
Alabama at Birmingham, Birmingham,
Alabama 35294-4400, USA

1. Introduction
Correspondence e-mail: [email protected] NAD+ is a ubiquitous and essential cofactor that is involved in
multiple oxidation–reduction reactions in all living cells (Berger et al.,
2004). NAD+ is biosynthesized de novo or through the pyridine-
Received 23 July 2008 salvage pathway; these two pathways converge at the penultimate
Accepted 10 September 2008 enzyme in NAD+ biosynthesis, nicotinic acid mononucleotide
adenylyltransferase (NaMNAT). Bacterial NaMNAT catalyzes the
PDB Reference: nicotinic acid mononucleotide
adenylyltransferase, 3dv2, r3dv2sf.
transfer of the adenylyl moiety of ATP to nicotinic acid mono-
nucleotide (NaMN) to form nicotinic acid adenine dinucleotide
(NaAD). NaAD is catalytically transformed to NAD+ by the enzyme
NAD+ synthetase. Since NaMNAT is a conserved enzyme and is
essential to the survival of every bacterium studied to date, it is
regarded as a potential target for the development of antibacterial
drugs.
The crystallization conditions for Bacillus anthracis NaMNAT (BA
NaMNAT) were optimized in part with the aid of self-interaction
chromatography (SIC), a technique which is growing in popularity for
its ability to identify and predict solution conditions that can improve
protein solubility (Tessier et al., 2002). The structure of BA NaMNAT
is described and compared with other deposited bacterial NaMNAT
structures.

2. Methods
2.1. Expression and purification of the BA NaMNAT nadD gene
The nadD gene (gi:9187226) was amplified from B. anthracis
genomic DNA. The sequence was inserted into expression vector
pET21b (Novagen Inc). This construction added a 12-residue tag to
the C-terminus of the recombinant protein with sequence LAAA-
LEHHHHHH; the formula weight of the protein was 23 347 Da. The
# 2008 International Union of Crystallography protein was overexpressed in Escherichia coli BL21 (DE3) pLysS
All rights reserved cells. The cells were grown at 310 K to an OD600 of 0.6 in Luria–

Acta Cryst. (2008). F64, 893–898 doi:10.1107/S1744309108029102 893

protein structure communications
Table 1 Table 2
Km and Vmax for BA NaMNAT. BA NaMNAT crystallographic data-collection and refinement statistics.
Substrate Km (mM) Vmax (mM min
1) Values in parentheses are for the outer shell.

NaMN 0.025  0.004 4.7  0.2 Data collection

ATP 0.044  0.003 4.3  0.1 Space group P21
Unit-cell parameters (Å,  ) a = 63.07, b = 100.59, c = 73.07,
 = 90,
= 110.42,  = 90
Matthews coefficient (Å3 Da
1) 2.33
Bertani medium containing 50 mg ml
1 ampicillin. Protein expression Solvent content (%) 47
Unique reflections 37491
was induced with 1 mM isopropyl
-d-1-thiogalactopyranoside Rmerge† 5.9 (28.5)
(IPTG) and growth took place for 4 h at 301 K after induction. Cells Redundancy 3.7 (3.3)
were harvested and purified at 277 K using affinity and size-exclusion Completeness (%) 97.6 (88.9)
I/(I) 10.5 (3.2)
chromatography (SEC) with Ni–NTA and Superdex 75 columns Refinement
(Amersham-Pharmacia). The SEC buffer composition optimized Resolution (Å) 50.0–2.3
with the aid of SIC was 50 mM Tris pH 7.0, 100 mM NaCl, 100 mM R factor/Rfree‡ (%) 0.228/0.263
No. of residues 751
Arg, 100 mM Glu, 1% glycerol, 1 mM DTT. The homogeneity of the No. of water molecules 131
purified protein was assessed by SDS–PAGE and dynamic light No. of sulfate ions 8
R.m.s. deviations from ideality
scattering. Bond lengths (Å) 0.007
Bond angles ( ) 1.2
2.2. Km determination Ramachandran plot, residues in
Most favored regions (%) 89.2
The Km values for ATP and NaMN were determined using a Additional allowed regions (%) 10.4
Generously allowed regions (%) 0.5
discontinuous HPLC assay. The reaction conditions were 50 mM Average B values (Å2)
HEPES pH 7.5, 10 mM MgCl2 and 0.25 mg ml
1 BA NAMNAT at Main-chain atoms 41.99
295 K. For determination of Km, ATP was varied from 6 to 600 mM Side-chain atoms 42.90
Solvent atoms 40.22
with NaMN held at 350 mM (14-fold Km) and NaMN was varied from
5 to 250 mM with ATP held at 600 mM (14-fold Km). The reaction was P P P P
† Rmerge = hkl i jIi ðhklÞ
hIðhklÞij= hkl i Ii ðhklÞ, where Ii(hkl) is the intensity of
stopped at various time points by the addition of 1.2 M guanidine– the ith measurement of reflection

hkl and

PhI(hkl)i is the average value over multiple
measurements. ‡ R factor =
jFo j
jFc j
= jFo j, where Fo and Fc are the observed and
HCl. The reactants and product were separated on a 4.6  100 mm calculated structure factors, respectively. Rfree was calculated for 5% of the reflections
Synergi Polar-RP column. The peak area of the product NaAD was removed randomly from the refinement.
used to calculate the production of NaAD. The reaction-progress
curves were linear under these conditions. The apparent initial
velocity V0 (mM min
1) was equivalent to the slope of the reaction- would improve protein solubility. SIC was also used to monitor
progress curve. V0 was plotted against the substrate concentration. crystallization optimization.
The Km and Vmax values were determined by a nonlinear fit of the
curve to the Michaelis–Menten equation using Origin software 2.4. Crystallization and X-ray data collection
(Microcal Software Inc., Northampton, Massachusetts, USA). The BA NaMNAT was concentrated to 4 mg ml
1 in SEC buffer
Km and Vmax reported error terms are the standard errors in the containing 200 mM trehalose. Initial crystallization screening was
calculated fit. carried out by the vapor-diffusion method at 297 K. 1 ml protein
solution mixed with 1 ml reservoir liquor was equilibrated against
2.3. Self-interaction chromatography (SIC) 1 ml reservoir screening solution in 24-well Nextal plates (Qiagen).
The second virial coefficient (B22) is an indicator of protein Bar-shaped crystals appeared within one week using Hampton
aggregation under certain conditions (George et al., 1997). SIC is an Crystal Screen kit I condition No. 39. Optimization was conducted
affinity-chromatography system used to measure B22 under a variety and the final refined reservoir condition was 2.1 M ammonium
of conditions; a description of the technique can be found in Tessier et sulfate, 100 mM HEPES pH 7.0, 2% PEG 400 and 10 mM MgCl2.
al. (2002). SIC was carried out for BA NaMNAT with of Arg, Glu or Crystals were soaked in artificial mother liquor containing 25%
trehalose additives in order to search for solution conditions that glycerol and flash-frozen before data collection. X-ray diffraction
data were collected on the Southeast Regional Collaborative Access
Team (SER-CAT) 22-ID beamline at the Advanced Photon Source,
Argonne National Laboratory using a MAR 300 CCD detector. A
total of 360 frames with 0.5 oscillation angle were collected at 100 K
using wavelength of 1 Å and a crystal-to-detector distance of 200 mm.
Data were processed using HKL-2000 (Otwinowski & Minor, 1997).

2.5. Structure determination and refinement

The structure of BA NaMNAT was determined by molecular
replacement using the MOLREP program in the CCP4 suite
(Collaborative Computational Project, Number 4, 1994). The struc-
ture of the B. subtilis NaMNAT (BS NaMNAT) dimer (PDB code
1kam) was used as a search model. The molecular-replacement
solution was further refined in the Crystallography & NMR System
Figure 1
Dimer structure of BA NaMNAT (chain A in green and chain B in cyan). (Brünger et al., 1998) and interactive sessions of model building were
Secondary structures are labelled for chain A. performed in Coot (Emsley & Cowtan, 2004). The structure was

894 Lu et al.  Nicotinic acid mononucleotide adenylyltransferase Acta Cryst. (2008). F64, 893–898
 protein structure communications
validated using the ADIT validation server (http://deposit.pdb.org/ additives in order to search for conditions to eliminate precipitation.
validate/; Yang et al., 2004) and deposited in the PDB under code As shown in Appendix A, Arg, Glu and trehalose improved the
3dv2. protein solubility. The final optimized gel-filtration buffer contained
100 mM Arg and 100 mM Glu. Trehalose was added at a concen-
tration of 200 mM during protein concentration and BA NaMNAT
3. Results could be concentrated to more than 12 mg ml
1 with no precipitation.
4 mg ml
1 protein was used for crystallization screening using the
3.1. Characterization and crystallization of NaMNAT sitting-drop and hanging-drop vapor-diffusion methods. Several
Purified B. anthracis NaMNAT exhibits the Km and Vmax values crystals with a variety of shapes were found in conditions from a 360-
shown in Table 1, which are similar to those found for BS NaMNAT condition in-house screen (Lisa screen360), as well as from Hampton
(Olland et al., 2002). The protein had an initial concentration of Crystal Screens. An SIC experiment was performed on the best hit,
2.7 mg ml
1 after nickel-affinity chromatography. However, after which was from Hampton Crystal Screen I condition No. 39. As
overnight dialysis against the SEC buffer (50 mM Tris–HCl pH 7.0, shown in Appendix A, the calculated B22 value of this condition
100 mM NaCl, 1% glycerol, 1 mM DTT) a precipitant was observed resided within the range of values known to be conducive to crys-
and only 0.4 mg ml
1 protein remained in solution. Differential tallization (George et al., 1997). This condition was then refined and
scanning calorimetry suggested that the protein was highly aggre- the final optimized crystallization condition was 2.1 M ammonium
gated. A second purification was performed: the nickel column was sulfate, 100 mM HEPES pH 7.0, 2% PEG 400 and 10 mM MgCl2.
followed by SEC. The latter was made possible by the incorporation Bar-shaped crystals grew to dimensions of 0.05  0.1  0.2 mm after
of 50 mM Arg and 50 mM Glu into the SEC buffer, which improved one week in the optimized condition.
the solubility. SIC experiments were performed on the protein A summary of the data-collection and refinement statistics is
obtained from SEC with various combinations and concentrations of shown in Table 2. The structure was validated and deposited in the

Figure 2
(a) Overlay of apo BA NaMNAT (PDB code 3dv2, green; 2qtm, blue), apo BS NaMNAT (1kam, yellow) and BS NaMNAT–NaAD complex (1kaq, red; NaAD is shown in
stick representation and colored by element). Secondary structures, N- and C-termini are labelled. (b–d) The three regions with the greatest conformational flexibility. Each
region is enlarged and rotated to show most clearly the differences in conformation between the proteins. All structures shown in (a) plus BA NaMNAT–NaAD (PDB code
2qtr; magenta) are included. The first and last residues of each region are labelled for BA.

Acta Cryst. (2008). F64, 893–898 Lu et al.  Nicotinic acid mononucleotide adenylyltransferase 895
protein structure communications
PDB with code 3dv2. During the course of the preparation of this -helices E and F. The C-terminal His-containing tag was not
manuscript, coordinates were released for the independently solved observed on any of the monomers in the crystal structure.
structure of apo BA NaMNAT (PDB code 2qtm) and two ligated
forms complexed with NMN (2qtn) and NXX (2qtr), respectively; the
latter ligand is apparently the product NaAD (V. C. Sershon, B. D. 3.3. Structural comparison with BS NaMNAT
Santarsiero & A. D. Mesecar, unpublished work). BA NaMNAT and BS NaMNAT share 56% sequence identity and
76% similarity. The present apo BA NaMNAT monomer structure
(PDB code 3dv2) is shown in Fig. 2(a) superimposed with apo BS
NaMNAT (1kam), the BS NaMNAT–NaAD complex (1kaq) and apo
3.2. Overall structure of BA NaMNAT BA NaMNAT (2qtm). The r.m.s.d.s of the respective C positions are
BA NaMNAT belongs to the  and
protein (/
) class. There are 1.13 Å (3dv2:2qtm) and 1.22 Å (3dv2:1kam), indicating high conser-
four molecules in the asymmetric unit, forming two dimers AB and vation of the protein structure between the two species, although it is
CD. The four molecules form a distorted tetrahedron in which the surprising that the r.m.s.d. between the two BA apo structures is not
primary dimer-to-dimer interface is between residues 135 and 139 lower. It can be seen from the superpositioned structures that there
from monomers A and C, which form a local pseudo-twofold are three regions of greater conformational diversity that are not only
symmetry axis. The dimer interfaces are stabilized by main-chain different from one apo structure to the next, but also change position
interactions between residues 151–153 from monomers A and B or C upon ligation, thus suggesting a degree of flexibility, viz. residues 40–
and D, which form distorted antiparallel
-strands with close 53 (between
-strands 2 and 3), residues 104–124 (between
-strands
hydrogen-bond contacts about a twofold-symmetric dimer axis. 4 and 5) and residues 132–143 (between
-strands 5 and 6). After
Reciprocal side-chain hydrogen bonds involved in holding the dimer removing the above three flexible regions, the C r.m.s.d.s were
together are Asn23–Thr168, Glu24–Arg162, Glu67–Lys170 and 0.71 Å (3dv2:2qtm) and 0.97 Å (3dv2:1kam).
Glu151–Arg133. These residues are mostly conserved between BA In Figs. 2(b), 2(c) and 2(d), these three respective regions are
and BS NaMNAT. Size-exclusion chromatography and dynamic light enlarged to show detail and the BA NaMNAT–NaAD complex (PDB
scattering also indicate that the protein is a dimer in solution (data code 2qtr) is included. These overlays as well as comparisons with
not shown). As expected, the dimer structure (Fig. 1) is very similar to other published NaMNAT structures lend support to the proposal
PDB entry 2qtm (V. C. Sershon, B. D. Santarsiero & A. D. Mesecar, that conformational diversity is an intrinsic property of the three
unpublished work) as well as to that observed for apo BS NaMNAT regions. Published NaMNAT structures derived from E. coli display
(PDB code 1kam; Olland et al., 2002). The monomer is composed of a flexibility in these regions dependent on the presence or absence of
classic Rossmann fold consisting of a right-handed
//
super ligand, while for Staphylococcus aureus there is no apo NaMNAT
secondary-structure element formed by six twisted parallel
-strands structure but two different NaAD structures derived from crystals
with order 321456 in the center flanked by -helices A and B on one exhibiting different space groups that exhibit different conformations
side and -helices C/C0 and D/D0 on the other side of the central in the N-terminal flexible region. In contrast, the Pseudomonas

-sheet. After the Rossmann fold is another domain composed of aeruginosa and human enzymes show little to no change upon liga-

Figure 3
Sequence alignment of NaNMATs from B. anthracis (BA), B. subtilis (BS), S. aureus (SA), E. coli (EC) and P. aeruginosa (PA). The structure of BA NaMNAT was used to
generate the structural annotations. The alignment was performed with ClustalW (Chenna et al., 2003) and the figure was prepared with ESPript (Gouet et al., 1999)

896 Lu et al.  Nicotinic acid mononucleotide adenylyltransferase Acta Cryst. (2008). F64, 893–898
 protein structure communications
tion in these regions. An alignment of bacterial NaMNAT sequences Table 3
is shown in Fig. 3. Experimental B22 values for Hampton Crystal Screen condition No. 39 (100 mM
HEPES pH 7.5, 2 M ammonium sulfate, 2% PEG 400).
The apo BS NaMNAT structure is the only published apo
NaMNAT structure that lacks electron density for the region The protein buffer was 50 mM Tris pH 7.0, 100 mM NaCl, 1% glycerol, 1 mM DTT,
100 mM Arg, 100 mM Glu and 200 mM trehalose. On increasing the percentage of the
containing residues 40–53, whereas in apo BA NaMNAT these resi- crystallization condition, the B22 values became more negative and closer to the
dues were easily modeled (Fig. 4); they are also present in apo BA ‘crystallization slot’.
NaMNAT (2qtm). In the BS NaMNAT–NaAD (1kaq) and BA Solution (%) B22  104 (mol ml g
2) Std dev.  104
NaMNAT–NaAD (2qtr) structures, the conserved 42PPHK45
sequence in this region is observed to interact with the nicotinic ring 1 12.5 3.8 0.61
2 25 2.8 0.75
of NaAD through Pro43 and His44 and is also involved in the 3 50
0.46 0.045
interaction with ATP through His44-Lys45 in the P. aeruginosa 4 75
0.95 0.075
NaMNAT–ATP complex structure (PDB code 1yun). When substrate
is bound, those residues are pulled in towards the binding site.
However, residues 40–53 of both apo BA NaMNAT structures are moves significantly toward the adenyl-diphosphate part of NaAD,
stabilized away from the substrate-binding site; this might be caused covering the binding site. In the BS and BA apo structures, the
by an interaction with a symmetry-related molecule as shown in Fig. 4. substrate-binding site is not covered by residues 132–143.
In 2qtm, which is derived from a different space group, a similar There are two sulfate ions in each monomer of apo BA NaMNAT
intermolecular interaction is made with a symmetry-related molecule. (3dv2) and they are located at the ATP-binding site of the protein.
It is also possible that this region is naturally stable and provided a The two sulfate ions form hydrogen bonds with residues Thr10,
crystal contact that enabled crystallization. This intermolecular His15, His18 and Arg160. In comparison, the three ATP phosphates
contact is not present in the crystals of apo BS NaMNAT. Residues in the PA NaMNAT–ATP complex form hydrogen bonds with Thr11,
47–50 involved in the contact exhibit high variation in sequence His16, His19 and Arg183. These sets of residues involved in hydrogen
comparisons. This could also contribute to the variation in confor- bonding to the sulfate ions and the ATP phosphates roughly super-
mation. pose onto each other and are conserved as shown in the sequence
Residues 102–124 in the apo BA NaMNAT structure have a alignment. However, it does not appear that the sulfate positions
conformation that is intermediate between the BS NaMNAT–NaAD correspond exactly to any of the phosphate positions. There is also a
complex and the apo BS NaMNAT structure. In the BS NaMNAT– water molecule found in all four monomers that bridges the two
NaAD complex this region is pulled close to the NaAD product. In sulfates together and hydrogen bonds to the main-chain N atoms of
the apo BS NaMNAT structure these residues reside far from the Ser156 and Ser157.
binding site. The conformation of residues 132–143 in apo BA
NaMNAT is almost the same as in the apo BS NaMNAT structure.
However, in the BS NaMNAT–NaAD complex structure this region 4. Discussion
B. anthracis is a Gram-positive spore-forming bacterial pathogen
which can cause anthrax and belongs to the class A priority bacterial
pathogens. The existence of strains resistant to present antibiotics
provides an incentive to develop new drug classes for treatment. The
present structure has been solved to facilitate the discovery of ther-
apeutics against this target.
The crystal structures of other NaMNAT/NMNATs (nicotinamide
mononucleotide adenylyltransferases) from E. coli, B. subtilis,
P. aeruginosa, S. aureus and Francisella tularensis (EC NaMNAT, BS
NaMNAT, PA NaMNAT, SA NaMNAT and FT NMNAT, respec-
tively) and two isoforms of human NMNAT termed PNAT-1 and
PNAT-3 have been resolved in various forms (Zhang et al., 2002;
Olland et al., 2002; Yoon et al., 2005; Han et al., 2006; Werner et al.,
2002; Garavaglia et al., 2002; Zhou et al., 2002; Huang et al., 2008).
Detailed comparative descriptions can be found in the citations
provided. These structures along with that of BA NaMNAT may be
useful to understand the differential binding of potential new inhi-
bitors.

APPENDIX A
Self-interaction chromatography (SIC) of BA NaMNAT
The solubility of BA NaMNATwas limited in Tris gel-filtration buffer.
SIC was performed to search for suitable additives to improve protein
Figure 4 solubility. Table 3 shows that 50/100 mM Arg and/or Glu and/or
Residues 43–51 in apo BA NaMNAT are shown in green with a 2Fo
Fc electron- trehalose affected the B22 values of protein solutions dramatically.
density map at 1 in blue. Part of a symmetry-related molecule is shown in yellow. The more positive the B22 value, the greater the protein solubility.
Crystal packing between the two chains is obvious and could account for the
stability of the loop 43–51. These residues are not visible in the BS apo structure, 100 mM Arg and 100 mM Glu were added to the optimized gel-
which does not have the same packing. filtration buffer and 200 mM trehalose was added during protein

Acta Cryst. (2008). F64, 893–898 Lu et al.  Nicotinic acid mononucleotide adenylyltransferase 897
protein structure communications
Table 4 the UAB Comprehensive Cancer Center Core (Support Grant P30
Experimental B22 values of the original gel-filtration buffer (50 mM Tris pH 7, CA-13148). Use of the Advanced Photon Source was supported by
100 mM NaCl, 10% glycerol, 1 mM DTT) with various additives.
the US Department of Energy, Office of Science, Office of Basic
Higher B22 values indicate better protein solubility. Energy Science under Contract No. W-31-109-Eng-38. This work was
B22  104 funded by NIH U01 AI 070386.
Solution (mol ml g
2) Std. dev.  104

1 Buffer only 0.24 0.54

2 Buffer + 50 mM Arg 7.0 1.2 References
3 Buffer + 100 mM Arg 11 0.24
4 Buffer + 50 mM Glu 9.7 0.67 Berger, F., Ramirez-Hernandez, M. H. & Ziegler, M. (2004). Trends Biochem.
5 Buffer + 100 mM Glu 6.1 1.9 Sci. 29, 111–118.
6 Buffer + 50 mM trehalose 0.83 0.46 Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-
7 Buffer + 100 mM trehalose 2.6 0.35 Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read,
8 Buffer + 50 mM Arg + 50 mM Glu 9.3 0.93 R. J., Rice, L. M., Simonson, T. & Warren, G. L. (1998). Acta Cryst. D54,
9 Buffer + 50 mM Arg + 50 mM trehalose 9.2 0.32
10 Buffer + 50 mM Glu + 50 mM trehalose 3.8 0.24
905–921.
11 Buffer + 50 mM Arg + 50 mM Glu + 14 0.53 Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. &
50 mM trehalose Thompson, J. D. (2003). Nucleic Acids Res. 31, 3497–3500.
Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50,
760–763.
Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132.
concentration. The protein solubility was increased from 0.3– Garavaglia, S., Dangelo, I., Emanuelli, M., Carnevali, F., Pierella, F., Magni, G.
0.4 mg ml
1 to more than 12 mg ml
1. & Rizzi, M. (2002). J. Biol. Chem. 277, 8524–8530.
The second virial coefficient (B22) has been shown to predict George, A., Chiang, Y., Guo, B., Arabshahi, A., Cai, Z. & Wilson, W. W.
(1997). Methods Enzymol. 276, 100–110.
protein-crystal growth (George et al., 1997). A good protein solution Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. (1999). Bioinformatics, 15,
for crystallization normally has a B22 value between
0.5 and 305–308.

8  10
4 mol ml g
2, the so-called ‘crystallization slot’. SIC was Han, S., Forman, M. D., Loulakis, P., Rosner, M. H., Xie, Z., Wang, H., Danley,
used to measure protein B22 values in our best crystal hit, Hampton D. E., Yuan, W., Schafer, J. & Xu, Z. (2006). J. Mol. Biol. 360, 814–825.
Huang, N., Sorci, L., Zhang, X., Brautigam, C. A., Li, X., Raffaelli, N., Magni,
Crystal Screen condition No. 39. Table 4 shows that with different G., Grishin, N. V., Osterman, A. L. & Zhang, H. (2008). Structure, 16,
dilutions of the crystallization solution from 12.5% to 75%, the B22 196–209.
value shifted to the crystallization slot. This confirmed that the Olland, A. M., Underwood, K. W., Czerwinski, R. M., Lo, M. C., Aulabaugh,
optimized condition was a good one for BA NaMNAT crystallization. A., Bard, J., Stahi, M. L., Somers, W. S., Sullivan, F. X. & Chopra, R. (2002).
J. Biol. Chem. 277, 3698–3707.
The authors would like to thank Ms Tasha Kane for protein Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326.
purification and crystallization and Dr Irina I. Protasevich is thanked Tessier, P. M., Lenhoff, A. M. & Sandler, S. I. (2002). Biophys. J. 82, 1620–1631.
Werner, E., Ziegler, M., Lerner, F., Schweiger, M. & Heinemann, U. (2002).
for purification advice. DNA sequencing was carried out by the
FEBS Lett. 516, 239–244.
CFAR DNA Sequencing Core at UAB with NIH CFAR Core grant Yang, H., Guranovic, V., Dutta, S., Feng, Z., Berman, H. M. & Westbrook, J. D.
P30A127767; mass spectrometry of proteins was carried out by the (2004). Acta Cryst. D60, 1833–1839.
Mass Spectrometry Shared Facility within the UAB Comprehensive Yoon, H. J., Kim, H. L., Mikami, B. & Suh, S. W. (2005). J. Mol. Biol. 351,
Cancer Center Core (Support Grant P30 CA-13148) using a Waters/ 258–265.
Zhang, H., Zhou, T., Kurnasov, O., Cheek, S., Grishin, N. V. & Osterman, A.
Micromass Q-Tof mass spectrometer (purchased from an NCRR (2002). Structure, 10, 69–79.
Shared Instrumentation grant; S10 RR13795). Some X-ray diffraction Zhou, T., Kurnasov, O., Tomchick, D. R., Binns, D. D., Grishin, N. V., Marquez,
was carried out by the X-ray Crystallography Shared Facility within V. E., Osterman, A. L. & Zhang, H. (2002). J. Biol. Chem. 277, 13148–13154.

898 Lu et al.  Nicotinic acid mononucleotide adenylyltransferase Acta Cryst. (2008). F64, 893–898