Structural Properties of CXCL4L1 and Its Recognition of the CXCR3 N-Terminus.

Chemokine CXCL4L1, a homologue of CXCL4, is a more potent antiangiogenic ligand. Its structural property is correlated with the downstream receptor binding. The two chemokines execute their functions by binding the receptors of CXCR3A and CXCR3B. The receptors differ by an extra 51-residue extension in the CXCR3B N-terminus. To understand the binding specificity, a GB1 protein scaffold was used to carry different CXCR3 extracellular elements, and artificial CXCL4 and CXCL4L1 monomers were engineered for the binding assay. We first characterized the molten globule property of CXCL4L1. The structural property causes the CXCL4L1 tetramer to dissociate into monomers in low concentrations, but native CXCL4 adopts a stable tetramer structure in solution. In the titration experiments, the combination of the CXCR3A N-terminus and receptor extracellular loop 2 provided moderate and comparable binding affinities to CXCL4 and CXCL4L1, while sulfation on the CXCR3A N-terminal tyrosine residues provided binding specificity. However, the CXCR3B N-terminal extension did not show significant enhancement in the binding of CXCL4 or CXCL4L1. This result indicates that the tendency to form a chemokine monomer and the binding affinity together contribute the high antiangiogenic activity of CXCL4L1.

N-terminal Backbone Pairing Shifts in CCL5-12AAA14 Dimer Interface: Structural Significance of the FAY Sequence.

CC-type chemokine ligand 5 (CCL5) has been known to regulate immune responses by mediating the chemotaxis of leukocytes. Depending on the environment, CCL5 forms different orders of oligomers to interact with targets and create functional diversity. A recent CCL5 trimer structure revealed that the N-terminal conversed F12-A13-Y14 (12FAY14) sequence is involved in CCL5 aggregation. The CCL5-12AAA14 mutant with two mutations had a deficiency in the formation of high-order oligomers. In the study, we clarify the respective roles of F12 and Y14 through NMR analysis and structural determination of the CCL5-12AAA14 mutant where F12 is involved in the dimer assembly and Y14 is involved in aggregation. The CCL5-12AAA14 structure contains a unique dimer packing. The backbone pairing shifts for one-residue in the N-terminal interface, when compared to the native CCL5 dimer. This difference creates a new structural orientation and leads to the conclusion that F12 confines the native CCL5 dimer configuration. Without F12 anchoring in the position, the interfacial backbone pairing is permitted to slide. Structural plasticity occurs in the N-terminal interaction. This is the first case to report this structural rearrangement through mutagenesis. The study provides a new idea for chemokine engineering and complements the understanding of CCL5 oligomerization and the role of the 12FAY14 sequence.

The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein-Protein Interaction.

Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the ß-barrel and the C-terminal helix bundle. The ß1-ß2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding.

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H2CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε-Cε-Nζ of Lys and Hδ-Cδ-Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H2CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H2CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.

Alternative C-terminal helix orientation alters chemokine function: structure of the anti-angiogenic chemokine, CXCL4L1.

BACKGROUND: CXCL4L1 is a highly potent anti-angiogenic and anti-tumor chemokine, and its structural information is unknown. RESULTS: CXCL4L1 x-ray structure is determined, and it reveals a previously unrecognized chemokine structure adopting a novel C-terminal helix conformation. CONCLUSION: The alternative helix conformation enhances the anti-angiogenic activity of CXCL4L1 by reducing the glycosaminoglycan binding ability. SIGNIFICANCE: Chemokine C-terminal helix orientation is critical in regulating their functions. Chemokines, a subfamily of cytokines, are small, secreted proteins that mediate a variety of biological processes. Various chemokines adopt remarkable conserved tertiary structure comprising an anti-parallel ß-sheet core domain followed by a C-terminal helix that packs onto the ß-sheet. The conserved structural feature has been considered critical for chemokine function, including binding to cell surface receptor. The recently isolated variant, CXCL4L1, is a homologue of CXCL4 chemokine (or platelet factor 4) with potent anti-angiogenic activity and differed only in three amino acid residues of P58L, K66E, and L67H. In this study we show by x-ray structural determination that CXCL4L1 adopts a previously unrecognized structure at its C terminus. The orientation of the C-terminal helix protrudes into the aqueous space to expose the entire helix. The alternative helix orientation modifies the overall chemokine shape and surface properties. The L67H mutation is mainly responsible for the swing-out effect of the helix, whereas mutations of P58L and K66E only act secondarily. This is the first observation that reports an open conformation of the C-terminal helix in a chemokine. This change leads to a decrease of its glycosaminoglycan binding properties and to an enhancement of its anti-angiogenic and anti-tumor effects. This unique structure is recent in evolution and has allowed CXCL4L1 to gain novel functional properties.

Correlations between membrane immersion depth, orientation, and salt-resistance of tryptophan-rich antimicrobial peptides.

The efficacies of many antimicrobial peptides are greatly reduced in the presence of high salt concentrations therefore limiting their development as pharmaceutical compounds. PEM-2-W5K/A9W, a short Trp-rich antimicrobial peptide developed based on the structural studies of PEM-2, has been shown to be highly active against various bacterial strains with less hemolytic activity. Here, correlations between membrane immersion depth, orientation, and salt-resistance of PEM-2 and PEM-2-W5K/A9W have been investigated via solution structure and paramagnetic resonance enhancement studies. The antimicrobial activities of PEM-2-W5K/A9W and PEM-2 against various bacterial and fungal strains including multidrug-resistant and clinical isolates under high salt conditions were tested. The activities of the salt-sensitive peptide PEM-2 were reduced and diminished at high salt concentrations, whereas the activities of PEM-2-W5K/A9W were less affected. The results indicated that the strong salt-resistance of PEM-2-W5K/A9W may arise from the peptide positioning itself deeply into microbial cell membranes and thus able to disrupt the membranes more efficiently.

A streamlined method for preparing split intein for NMR study.

A protein ligase, intein, mediates a protein-splicing reaction. It can be split into two complementary fragments and reconstituted as a whole intein scaffold to perform protein trans-splicing. To understand the association of intein fragments and the splicing mechanism, it is necessary to produce a large quantity of split intein for structural study. Conventionally, two fragments are prepared separately and assembled in solution, but severe aggregation of intein fragments occurs, and precise control of the relative concentration of each fragment is difficult. Here, we present a streamlined method to incorporate a circular permutation concept into the production of split intein. By circular permutation of the native split Nostoc punctiforme DnaE intein (NpuInt), a new backbone opening is relocated to the native split site at residue 102. As the protein splicing activity is preserved, the expressed NpuInt can immediately self-cleave into a two-piece split NpuInt. Because of a tight association between the two complementary fragments, split NpuInt can be purified in one step. The idea is simple and applicable to other split inteins. Employing the new preparation, we use NMR spectra to assign the backbone and side chain resonances for the native split NpuInt.

Detection of a ternary complex of NF-kappaB and IkappaBalpha with DNA provides insights into how IkappaBalpha removes NF-kappaB from transcription sites.

It has been axiomatic in the field of NF-κB signaling that the formation of a stable complex between NF-κB and the ankyrin repeat protein IκBα precludes the interaction of NF-κB with DNA. Contradicting this assumption, we present stopped-flow fluorescence and NMR experiments that give unequivocal evidence for the presence of a ternary DNA-NF-κB-IκBα complex in solution. Stepwise addition of a DNA fragment containing the κB binding sequence to the IκBα-NF-κB complex results in changes in the IκBα NMR spectrum that are consistent with dissociation of the region rich in proline, glutamate, serine, and threonine (PEST) and C-terminal ankyrin repeat sequences of IκBα from the complex. However, even at high concentrations of DNA, IκBα remains associated with NF-κB, indicated by the absence of resonances of the free N-terminal ankyrin repeats of IκBα. The IκBα-mediated release of NF-κB from its DNA-bound state may be envisioned as the reverse of this process. The initial step would consist of the coupled folding and binding of the intrinsically disordered nuclear localization sequence of the p65 subunit of NF-κB to the well-structured N-terminal ankyrin repeats of IκBα. Subsequently the poorly folded C-terminal ankyrin repeats of IκBα would fold upon binding to the p50 and p65 dimerization domains of NF-κB, permitting the negatively charged C-terminal PEST sequence of IκBα to displace the bound DNA through a process of local mass action.

Significance of heparin binding to basic residues in homologous to the amino terminus of hepatoma-derived growth factor and related proteins.

Hepatoma-derived growth factor (HDGF) recognizes cell surface heparan sulfate to promote its internalization though binding to its N-terminal HATH (homologous to amino terminus of HDGF) domain. HDGF-related proteins (HRPs) all have the HATH domain in their N terminus. In this study, we report on the commonality of heparin binding in all HRPs with a broad range of heparin-binding affinity: HRP-4 is the strongest binder, and the lens epithelium-derived growth factor shows a relatively weak binding, with binding affinities (K(D)) showing 30-fold difference in magnitude. With the HDGF HATH domain used as a model, residue K19 was the most critical basic residue in molecular recognition and protein internalization, and with its proximal proline-tryptophan-tryptophan-proline motif, coordinated a conformational change when binding to the heparin fragment. Other basic residues, K21, K61, K70, K72 and R79, confer added contribution in binding that the total ionic interaction from these residues represents more than 70% of the binding energy. Because the positive-charged residues are conserved in all HRP HATH domains, heparin binding outside of cells might be of equal importance for all HRPs in mediating downstream signaling; however, distinct effects and/or distribution might be associated with the varying affinities to heparin.

CheShift-2 resolves a local inconsistency between two X-ray crystal structures.

Since chemical shifts provide important and relatively accessible information about protein structure in solution, a Web server, CheShift-2, was developed for structure interrogation, based on a quantum mechanics database of (13)C( α ) chemical shifts. We report the application of CheShift-2 to a local inconsistency between two X-ray crystal structures (PDB IDs 1IKN and 1NFI) of the complex between the p65/p50 heterodimer of NFκB and its inhibitor IκBα. The availability of NMR resonance assignments that included the region of the inconsistency provided an opportunity for independent validation of the CheShift-2 server. Application of the server showed that the (13)C( α ) chemical shifts measured for the Gly270-Pro281 sequence close to the C-terminus of IκBα were unequivocally consistent with the backbone structure modeled in the 1IKN structure, and were inconsistent with the 1NFI structure. Previous NOE measurements had demonstrated that the position of a tryptophan ring in the region immediately N-terminal in this region was not consistent with either structure. Subsequent recalculation of the local structure in this region, based on the electron density of the deposited structure factors for 1IKN, confirmed that the local backbone structure was best modeled by 1IKN, but that the rotamer of Trp258 is consistent with the 1NFI structure, including the presence of a hydrogen bond between the ring NεH of Trp258 and the backbone carbonyl group of Gln278. The consensus between all of these measures suggests that the CheShift-2 server operates well under circumstances in which backbone chemical shifts are available but where local plasticity may render X-ray structural data ambiguous.

Cell surface heparan sulfates mediate internalization of the PWWP/HATH domain of HDGF via macropinocytosis to fine-tune cell signalling processes involved in fibroblast cell migration.

HDGF (hepatoma-derived growth factor) stimulates cell proliferation by functioning on both sides of the plasma membrane as a ligand for membrane receptor binding to trigger cell signalling and as a stimulator for DNA synthesis in the nucleus. Although HDGF was initially identified as a secretory heparin-binding protein, the biological significance of its heparin-binding ability remains to be determined. In the present study we demonstrate that cells devoid of surface HS (heparan sulfate) were unable to internalize HDGF, HATH (N-terminal domain of HDGF consisting of amino acid residues 1-100, including the PWWP motif) and HATH(K96A) (single-site mutant form of HATH devoid of receptor binding activity), suggesting that the binding of HATH to surface HS is important for HDGF internalization. We further demonstrate that both HATH and HATH(K96A) could be internalized through macropinocytosis after binding to the cell surface HS. Interestingly, HS-mediated HATH(K96A) internalization is found to exhibit an inhibitory effect on cell migration and proliferation in contrast with that observed for HATH action on NIH 3T3 cells, suggesting that HDGF exploits the innate properties of both cell surface HS and membrane receptor via the HATH domain to affect related cell signalling processes. The present study indicates that MAPK (mitogen-activated protein kinase) signalling pathways could be affected by the HS-mediated HATH internalization to regulate cell migration in NIH 3T3 fibroblasts, as judged from the differential effect of HATH and HATH(K96A) treatment on the expression level of matrix metalloproteases.

High Level Expression and Purification of Cecropin-like Antimicrobial Peptides in Escherichia coli.

Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34-39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.

Anti-apoptotic BCL-2 regulation by changes in dynamics of its long unstructured loop.

BCL-2, a key protein in inhibiting apoptosis, has a 65-residue-long highly flexible loop domain (FLD) located on the opposite side of its ligand-binding groove. In vivo phosphorylation of the FLD enhances the affinity of BCL-2 for pro-apoptotic ligands, and consequently anti-apoptotic activity. However, it remains unknown as to how the faraway, unstructured FLD modulates the affinity. Here we investigate the protein-ligand interactions by fluorescence techniques and monitor protein dynamics by DEER and NMR spectroscopy tools. We show that phosphomimetic mutations on the FLD lead to a reduction in structural flexibility, hence promoting ligand access to the groove. The bound pro-apoptotic ligands can be displaced by the BCL-2-selective inhibitor ABT-199 efficiently, and thus released to trigger apoptosis. We show that changes in structural flexibility on an unstructured loop can activate an allosteric protein that is otherwise structurally inactive.

Type IIb Heat Labile Enterotoxin B Subunit as a Mucosal Adjuvant to Enhance Protective Immunity against H5N1 Avian Influenza Viruses.

Human infections with highly pathogenic avian influenza H5N1 viruses persist as a major global health concern. Vaccination remains the primary protective strategy against H5N1 and other novel avian influenza virus infections. We investigated the use of E. coli type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant for intranasal immunizations with recombinant HA proteins against H5N1 avian influenza viruses. Use of LTIIb-B5 adjuvant elicited more potent IgG, IgA, and neutralizing antibody titers in both sera and bronchoalveolar lavage fluids, thus increasing protection against lethal virus challenges. LTIIb-B5 mucosal adjuvanticity was found to trigger stronger Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Studies of anti-IL-17A monoclonal antibody depletion and IL-17A knockout mice also suggest the contribution from Th17 cellular response to anti-H5N1 protective immunity. Our results indicate a link between improved protection against H5N1 live virus challenges and increased Th17 response due to the use of LTIIb-B5 mucosal adjuvant with HA subunit proteins.

Integrative Model to Coordinate the Oligomerization and Aggregation Mechanisms of CCL5.

CC-type chemokine ligand 5 (CCL5) is involved in the pathogenesis of many inflammatory conditions. Under physiological conditions, CCL5 oligomerization and aggregation are considered to be responsible for its inflammatory properties. The structural basis of CCL5 oligomerization remains controversial because the current oligomer models contain no consensus interactions. In this study, NMR and biophysical analyses proposed evidence that the CC-type CCL5 dimer acts as the basic unit to constitute the oligomer and that CCL5 oligomerizes alternatively through E66-K25 and E66-R44/K45 interactions. In addition, a newly determined trimer structure, constituted by CCL5 and the E66S mutant, reported an interfacial interaction through the N-terminal 12FAY14 sequence. The interaction contributes to CCL5 aggregation and precipitation but not to oligomerization. In accordance with the observations, an integrative model explains the CCL5 oligomerization and aggregation mechanism in which CCL5 assembly consists of two types of dimer-dimer interactions and one aggregation mechanism. For full-length CCL5, the molecular accumulation triggers oligomerization through the E66-K25 and E66-R44/K45 interactions, and the 12FAY14 interaction acts as a secondary effect to derive aggregation and precipitation. In contrast, the E66-R44/K45 interaction might dominate in CCL5 N-terminal truncations, and the interaction would lead to the filament-like formation in solution.

Functional dynamics of the folded ankyrin repeats of I kappa B alpha revealed by nuclear magnetic resonance.

Inhibition of nuclear factor kappaB (NF-kappaB) is mainly accomplished by IkappaB alpha, which consists of a signal response sequence at the N-terminus, a six-ankyrin repeat domain (ARD) that binds NF-kappaB, and a C-terminal PEST sequence. Previous studies with the ARD revealed that the fifth and sixth repeats are only partially folded in the absence of NF-kappaB. Here we report NMR studies of a truncated version of IkappaB alpha, containing only the first four ankyrin repeats, IkappaB alpha(67-206). This four-repeat segment is well-structured in the free state, enabling full resonance assignments to be made. H-D exchange, backbone dynamics, and residual dipolar coupling (RDC) experiments reveal regions of flexibility. In addition, regions consistent with the presence of micro- to millisecond motions occur periodically throughout the repeat structure. Comparison of the RDCs with the crystal structure gave only moderate agreement, but an ensemble of structures generated by accelerated molecular dynamics gave much better agreement with the measured RDCs. The regions showing flexibility correspond to those implicated in entropic compensation for the loss of flexibility in ankyrin repeats 5 and 6 upon binding to NF-kappaB. The regions showing micro- to millisecond motions in the free protein are the ends of the beta-hairpins that directly interact with NF-kappaB in the complex.

Salt-sensitive intein for large-scale polypeptide production.

In this chapter, we propose to use salt-sensitive intein as a fusion protein to promote polypeptide expression; the removal of intein from the target sequence requires no enzyme, only a buffer change. The method will be particularly helpful for large-scale polypeptide preparations. Intein is an enzyme that can perform N- and C-terminal self-cleavage. Upon introduction of a mutation to eliminate the N-terminal cleavage activity, the C-terminal cleavage function can still be preserved. This feature was used to develop intein as a fusion protein through conjugation with a given sequence to promote protein expression in a biosynthesis system. Fused intein could later be separated from the target sequence through a C-terminal self-cleavage reaction. Here, a type of salt-sensitive intein is characterized in which ionic strength becomes an effector to control the self-cleavage activity. Low salt concentrations favor the cleavage reaction. Thus, using salt-sensitive intein as a fusion protein simply requires a buffer change to activate the self-cleavage mechanism, which makes it an enzyme-free process. This process has many advantages, including low cost, no extra residue remaining after cleavage, feasibility for preparing proteins starting from a non-Met codon and a special benefit for producing isotope-labeled peptides.

Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells.

The delivery of active proteins into cells (protein transfection) for biological purposes offers considerable potential for clinical applications. Herein we demonstrate that, with a readily available, inexpensive organic agent, the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) method can be used for simple and efficient protein transfection. By mixing proteins with a pure HEPES solution before they are applied to live cells, proteins with various molecular weights (including antibodies, recombinant proteins, and peptides) were successfully delivered into the cytoplasm of different cell types. The protein transfection efficiency of the HEPES method was not inferior to that of commercially available systems that are both more expensive and time consuming. Studies using endocytotic inhibitors and endosomal markers have revealed that cells internalize HEPES-protein mixtures through endocytosis. Results that HEPES-protein mixtures exhibited a low diffusion coefficient suggest that HEPES might neutralize the charges of proteins and, thus, facilitate their cellular internalization. Upon internalization, the cytosolic antibodies caused the degradation of targeted proteins in TRIM21-expressing cells. In summary, the HEPES method is efficient for protein transfection and has potential for myriad clinical applications.

PWWP module of human hepatoma-derived growth factor forms a domain-swapped dimer with much higher affinity for heparin.

Hepatoma-derived growth factor (hHDGF)-related proteins (HRPs) comprise a new growth factor family sharing a highly conserved and ordered N-terminal PWWP module (residues 1-100, previously referred to as a HATH domain) and a variable disordered C-terminal domain. We have shown that the PWWP module is responsible for heparin binding and have solved its structure in solution. Here, we show that under physiological conditions, both the PWWP module and hHDGF can form dimers. Surface plasmon resonance (SPR) studies revealed that the PWWP dimer binds to heparin with affinity that is two orders of magnitude higher (K(d)=13 nM) than that of the monomeric PWWP module (K(d)=1.2 microM). The monomer-dimer equilibrium properties and NMR structural data together suggest that the PWWP dimer is formed through a domain-swapping mechanism. The domain-swapped PWWP dimer structures were calculated on the basis of the NMR data. The results show that the two PWWP protomers exchange their N-terminal hairpin to form a domain-swapped dimer. The two monomers in a dimer are linked by the long flexible L2 loops, a feature supported by NMR relaxation data for the monomer and dimer. The enhanced heparin-binding affinity of the dimer can be rationalized in the framework of the dimer structure.

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA.

Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248-365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248-280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248-280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.