Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science5, 6A/2004G344

Crystal Structures of Shark Immunoglobulin New Antigen Receptors (IgNARs)
1Victor A. Streltsov*1,2, Jose N. Varghese1 and Stewart D. Nuttall1,2
CSIRO HSN and 2CRC for Diagnostics, 343 Royal Parade, Parkville, Vic 3052, Australia

Introduction
Immunoglobulin new antigen receptors (IgNARs) are aunique antibody isotype found in the serum of sharks[1,2]. They are bivalent, but target antigen through asingle immunoglobulin variable domain (~13kDa)
displaying two complementarity determining region(CDR) loops. In contrast, conventional antibodies have avariable heavy (VH) and light (VL) domains format(~26kDa) and bind antigen through up to six CDRs. To
compensate for their reduced size, IgNARs encode
unusually long and structurally complex CDR-3s, whichdisplay a high degree of variability.
To date, three IgNAR isotypes have been identified,
which vary in the number and configuration of theirframework cysteine residues, and time of appearance inshark development. Type 3 IgNARs, the last discovered,display core sequence similarity to type 2 and limited
diversity in both the size and composition of their CDR
loop regions. They appear early in shark development
prior to maturation of the full adaptive immune response.
Both type 1 and -2 IgNAR levels increase as the shark
immune system is exposed to exogenous antigen, andshow significant diversity consistent with extensive
antibody affinity maturation.
Recently, both our laboratory [3] and [4] have reported
crystallographic structures for IgNAR variable domains(type 2 and 1, respectively), which provide significantinsight into their evolutionary origin and antigen-binding
strategy. Interestingly, the IgNAR immunoglobulin foldresembles I-set proteins (eg cell adhesion molecules) as
much as it does conventional V-set immunoglobulins (egVH/VL antibodies; T-cell receptors), suggesting an early
divergence amongst the molecules of the shark immunesystem [5]. The structures also clearly delineate the type 1
and type 2 isotypes. For type 2, a disulphide bridge
usually, though not in our first structures, links the CDR1and CDR3 regions producing a loop structure extending
high above the immunoglobulin framework. In contrast,
for type 1 two conserved framework cysteine residues
form disulphide bridges with matching residues within
the extended CDR3, distending the loop laterally. These
appear to be two related strategies to enhance stability,
and concurrently position the extended loop allowing
access to cleft-like epitopes, such as the lysozyme active
site in one of the reported structures [4], in a manner
similar to that observed in camelid VHHs, the only othernaturally occurring single domain antibodies.
Now, we have obtained X-ray data and solved thestructure of a fully natural type 2 IgNAR variable domain.

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Users' Report

Experimental
X-ray diffraction data for small crystal of type 2IgNAR was collected at the multipole wiggler beamline
BL5. BL5 is equipped with collimating mirror, doublecrystal Si(111) monochromator and focusing mirror
which focuses X-rays to the ADSC Quantum 315 CCD
detector. Data were collected at -160ºC; the crystals
required no added cryoprotectant. The data processing
was carried out using the DENZO/SCALEPACK suite.
Diffraction data statistics are summarized in below.
Space Group
Unit cell (Å) (a,b,c)
Resolution (Å)
Wavelength (Å)
Unique reflections
Multiplicity
Completeness (%)
I/σ(I)
2
χ
Rmerge (%)

P2121238.27,68.32,39.51
2.1
1.000
6048
7.2
98.6
15.2
1.03
12.6

Results and Discussion
The structure of type 2 IgNAR was determined bymolecular replacement using CCP4 MOLREP. The searchmodel was the IgNAR structure [3] without the CDR3
loop. The final refinement included converged to R/Rfreevalues of 0.217/0.280, respectively, for data up to 2.1 Åresolution. The coordinates have been deposited in theProtein Data Bank under the accession code 2COQ. Thestructure of a fully natural type 2 IgNAR variable domain
possesses a disulphide bridge linking the CDR1 and -3
loops and the complete structure analysis will be
published elsewhere [6].
References[1] A. S. Greenberg, D . Avila, M . Hughes, A . Hughes, E.
McKinney, M. F. Flajnik. Nature, 374, 168 (1995).
[2] S. D. Nuttall, U. V. Krishnan, M. Hattarki, R . De Gori,R. A. Irving, P. J. Hudson, Mol Immunol, 38,313 (2001).
[3] V. A. Streltsov, J. N. Varghese, J. A. Carmichael, R. A.
Irving, P. J. Hudson, S. D. Nuttall, Proc Natl Acad Sci
USA, 101, 12444 (2004).
[4] R. L. Stanfield, H. Dooley, M. F. Flajnik, I. A. Wilson.
Science, 305, 1770 (2004).
[5] V. A. Streltsov, S. D. Nuttall, Immunology Letters
97/1, 159 (2004).
[6] V. A. Streltsov, J. A. Carmichael, S. D. Nuttall in
preparation.
* victor.streltsov@csiro.au