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   GTP-binding proteins of relative molecular mass 67,000 with a
   high-turnover GTPase activity^[29]3 and an antiviral effect^[30]4. Here
   we have determined the crystal structure of full-length human GBP1 to
   1.8 Å resolution. The amino-terminal 278 residues constitute a modified
   G domain with a number of insertions compared to the canonical Ras
   structure, and the carboxy-terminal part is an extended helical domain
   with unique features. From the structure and biochemical experiments
   reported here, GBP1 appears to belong to the group of large GTP-binding
   proteins that includes Mx and dynamin, the common property of which is
   the ability to undergo oligomerization with a high
   concentration-dependent GTPase activity^[31]5.

   Guanylate-binding proteins (GBP1 and 2) were originally identified as
   proteins from an extract of human fibroblasts treated with interferons,
   gamma -interferon being the most effective, that bind to agarose-bound
   GMP, GDP and GTP^[32]1, ^[33]2. Smaller guanylate-binding proteins of
   relative molecular mass 47,000 (M[r] = 47K)^[34]6 are also induced by
   gamma -interferon, whereas alpha - and beta -interferon induce
   antiviral GTP-binding Mx proteins^[35]7. Human (h)GBP1 is expressed to
   mediate an antiviral effect against vesicular stomatitis virus and
   encephalomyocarditis virus^[36]4. The biochemical properties of GBPs
   are clearly different from those of Ras-like and heterotrimeric
   GTP-binding proteins. They bind guanine nucleotides with low affinity
   (micromolar range), are stable in their absence and have a high
   turnover GTPase^[37]3, ^[38]8. In addition to binding GDP/GTP, they
   have the unique ability to bind GMP with equal affinity and hydrolyse
   GTP not only to GDP but also to GMP^[39]3. As a first step towards
   understanding the biochemistry and biology of GBPs, we have determined
   the three-dimensional structure of hGBP1. Furthermore, we show
   nucleotide-dependent oligomerization of hGBP1 and concentration
   dependence of its GTPase reaction rate.

   We determined the structure of full-length, histidine(His[6])-tagged
   hGBP1 in the absence of nucleotide to 1.8 Å. The final model comprises
   residues 6–583 and 341 water molecules ([40]Fig. 1a, [41]b), with some
   poorly defined, probably mobile loops (dashed lines). The
   crystallographic data are summarized in [42]Table 1. The structure can
   be divided into a compact, globular alpha , beta -domain (6–278), which
   we term the LG (Large G) domain, and an elongated, purely alpha
   -helical domain. The domains are connected by a short intermediate
   region consisting of one alpha -helix and a short two-stranded beta
   -sheet. The connecting region is not an independent domain; it is
   packed, via helix alpha 6, against the beta 1/ alpha 1 region of the LG
   domain, away from the presumed nucleotide-binding site (see below). It
   could be involved in stabilizing the relative location of the two
   domains against each other. The helical domain is composed of seven
   helices, which extend 90 Å away from the LG domain.

 §5§ [43] Figure 1: Primary, secondary and tertiary structure of hGBP1. §5§

   [44]Figure 1 : Primary, secondary and tertiary structure of hGBP1.
   Unfortunately we are unable to provide accessible alternative text for
   this. If you require assistance to access this image, or to obtain a
   text description, please contact npg@nature.com

   a, A model of the tertiary structure of hGBP1 presented as a ribbon,
   where the LG domain is in purple, the connecting region in green, the
   helical domain in yellow and alpha 12/ alpha 13 in cyan. Insertions,
   marked I2–I5 in b, are in violet. Dashed lines indicate disordered
   regions in the molecule. The tentative nucleotide-binding area,
   identified by a Ras–GBP overlay, is indicated by a sphere with radius
   7 Å. The topology is shown schematically using the same colour code. b,
   Sequence alignment of hGBP1 (Swissprot accession no. P32455) with Mag-2
   (EMBL acccession no. M81128) from mouse and chicken GBP1 (EMBL
   accession no. X92112), with the secondary structure assignment as
   determined using the programme DSSP^[45]29, with the same colour code
   as in a, and aligned with the secondary structure of Ras dot
   GppNHp^[46]30 (light blue lines). Contacts between helix alpha 12 and
   the rest of the protein are indicated: asterisk, direct; circle,
   water-mediated polar interactions; hash sign for both. For brevity,
   sequences with lowest homology have been chosen.
   [47]High resolution image and legend (233K)

 §5§ [48] Table 1: Crystallographic data statistics §5§

   [49]Table 1 - Crystallographic data statistics

   [50]Full table

   The LG domain of GBPs contains the conserved sequence elements of
   GTP-binding proteins with modifications. Originally the N/TKxD motif
   was believed to be absent in the GBPs^[51]2. An Asp-Asn mutation can
   produce a change in specificity from guanine to xanthine nucleotides in
   many GTP-binding proteins such as EF-Tu^[52]9. As an Asp-Asn mutation
   in the ^181TLRD^184 motif of hGBP1 behaves similarly, it is postulated
   that Asp 184 should bind the guanine base through a bidentate hydrogen
   bond^[53]8. It was thus expected that GBP1 would contain the Ras G
   domain or a variation thereof. The structure shows that the 278-residue
   globular domain largely resembles the canonical architecture of Ras (
   approx 170 residues), allowing for additions and insertions (labelled
   I). Superimposing the structures of hGBP1 and Ras-GDP ([54]Fig. 2a)
   gives a root mean square (r.m.s) deviation of 1.1 Å for 112 common C
   alpha atoms. The LG domain consists of an eight-stranded beta -sheet
   with six parallel and two anti-parallel strands surrounded by nine
   helices, whereas Ras contains six beta -strands and five helices
   ([55]Fig. 1b). Using the secondary structure elements of Ras as the
   basis for the comparison and retaining the corresponding numbering, the
   additional elements are beta 0, alpha 0 and beta -1 on the N-terminal
   side of the sheet, and helices alpha 3' (I3) and alpha 4' (I4)
   ([56]Fig. 1b). Apart from a short insertion (I1) in switch I, there are
   comparatively long loop insertions between the ^97DxxG^100 motif and
   alpha 2 (I2), and between beta 6 and alpha 5 (I5) of the canonical G
   domain, respectively ([57]Figs 1 and [58]2). Whereas I1, I2 and I5
   could be involved in nucleotide binding, I3 and I4, on the opposite
   side of the protein, apparently mediate contact with the C-terminal
   helix.

 §5§ [59] Figure 2: Comparison of hGBP1 and Ras structures. §5§

   [60]Figure 2 : Comparison of hGBP1 and Ras structures. Unfortunately we
   are unable to provide accessible alternative text for this. If you
   require assistance to access this image, or to obtain a text
   description, please contact npg@nature.com

   a, Superimposition of the LG domain of hGBP1 with the G domain of Ras
   in complex with GDP(PDB accession no. 1Q21) as a stereo view.
   N-terminal residues 1–36 of hGBP1 up to beta 1 have been omitted for
   clarity. The colour code is as in [61]Fig. 1; Ras is in cyan. b,
   Putative location of nucleotide-binding site in hGBP1. The regions of
   hGBP1 potentially involved in binding the guanine nucleotide are shown
   as obtained from a structural superimposition of RasGDP (in cyan) with
   the corresponding regions in hGBP1 (purple), highlighting functionally
   important residues necessary for binding and conformational change as
   balls or in ball-and-stick. Whereas Gly 60^ras overlays very well with
   Gly 100^hGBP1, residues D119/D184 and T35/T75 do not.
   [62]High resolution image and legend (79K)

   As GBP is stable in the absence of nucleotide, whereas Ras-like and G
   alpha GTP-binding proteins are not, it was of interest to investigate
   the effect of the absence of nucleotide on the structure. As all
   P-loop-containing proteins^[63]10 bind the beta / gamma -phosphate of
   the nucleotide in a similar manner, and as the role of Asp 184 in
   binding the guanine base is similar to that of the Asp of the canonical
   N/TKxD motif, we can locate the nucleotide-binding site of hGBP1 using
   the RasGDP–hGBP1 overlay ([64]Fig. 2b). From this comparison we can
   also see that, although part of the binding site is more accessible to
   the solvent than in Ras–nucleotide complexes, part of the polypeptide
   chain is in a position that interferes with nucleotide binding. Perhaps
   owing to the absence of nucleotide, the polypeptide chain around the
   binding site is mobile, as no electron density is visible for residues
   69–72 (I1) in the region analogous to switch I, residues 190–193 close
   to the ^181TLRD^184 motif and residues 244–257 in I5, close to the
   SAK/L motif, which is conserved only in the Ras family and is absent in
   GBPs.

   The (phosphate-binding) P loop^[65]10, residues 45–52, adopts a
   structure different from that of the Ras–nucleotide complexes. The
   invariant lysine residue of the P loop does not interact with the
   main-chain carbonyls for stabilization. Instead, in hGBP1 the loop is
   stabilized by interactions with the region analogous to switch II,
   involving hydrogen bonds between Tyr 47 (backbone N) and Asp 103,
   Lys 51 and Thr 98. Furthermore, the structure is not suited for
   nucleotide binding as the phosphates would clash with Tyr 47. The
   region corresponding to switch I in Ras is disordered in hGBP1. Thr 75
   appears to be analogous to Thr 35 in Ras, but is 5 Å away in the
   overlay ([66]Fig. 2b). The ^97DxxG^100 motif of hGBP1 superimposes well
   with that of switch II in Ras. D184 is 6 Å away from the corresponding
   D119 of the canonical N/TKxD motif and would have to move accordingly
   to occupy a similar position in the nucleotide-bound form. In general,
   it appears that the guanine nucleotide-binding site is partly open
   ([67]Fig. 2a, [68]b) such that the incoming nucleotide would enter the
   binding site base first and would then, after a corresponding
   conformational change, bind into the phosphate-binding area, as
   suggested for Ras by the structure of the Ras–Sos complex^[69]11.

   Ras proteins have an intrinsic GTPase reaction rate in the order of
   0-001–0.1 min^-1, whereas hGBP1 (ref. [70]8) has a rate of up to
   80 min^-1 (see below). The catalytic mechanism for Ras and G alpha
   proteins involves a glutamine (Gln 61 in Ras) that stabilizes the
   transition state and is itself stabilized by GAP in the GAP-catalysed
   mechanism^[71]12. As Gly 60^ras and Gly 100^hgbp1 are very close to
   each other ([72]Fig. 2b), we would predict that Gln 61 of Ras is
   structurally homologous to Leu 101 in hGBP1. Considering the importance
   of Gln 61 in the intrinsic and GAP-catalysed GTPase reaction of Ras, we
   conclude that the chemical mechanism of hydrolysis of GTP by hGBP1 is
   different from that of Ras. A similar hydrophobic residue corresponding
   to Gln 61 of Ras has also been identified in dynamins and Mx proteins.
   Discounting the influence of oligomerization on the rate of the GTPase
   reaction, only insertion I2 following Leu 101 appears to be close
   enough to the phosphate-binding site to be involved in catalysis. This
   ^103DxEKGD^108 motif is conserved in GBPs and contains charged residues
   that could have a catalytic role, either similar to that of the Arg in
   G alpha proteins or GAPs^[73]12 or as a catalytic base.

   The helical domain of hGBP1 can be considered to consist of two
   three-helix bundles, formed by helices alpha 7, alpha 8, alpha 9
   (residues 311–403) and helices alpha 9, alpha 10, alpha 11 (404–482),
   where helix alpha 9 is common to both ([74]Fig. 1a). Adjacent to and
   covering these two bundles is a very long helix alpha 12 that reaches
   back to the LG domain. This helix, which is predicted from the sequence
   to be a coiled-coil structure, is 78 residues long and stretches over
   118Å. At its C-terminal end there is a helical turn leading into
   another short helix, alpha 13, which makes a coiled-coil type of
   interaction with alpha 12. The last eight residues, including the
   prenylation-recognition Caax motif, not modified owing to recombinant
   expression in E. coli, are not visible in the structure.

   The two three-helix bundles give the molecule an elongated shape. The
   core of the helical domains is formed by hydrophobic residues, whereas
   the charged amino acids are exposed towards but interact only weakly
   with the residues from the long helix alpha 12. Thus, alpha 12 has
   almost no contact with the second (more remote) helical bundle (a
   single hydrogen bond between Glu 490 and Tyr 433, [75]Fig. 1b) whereas
   it forms seven direct side-chain and nine water-mediated contacts with
   the first. It is stabilized further by four direct and eight
   water-mediated contacts with insertions I3 and I4 ([76]Fig. 1) of the
   LG domain. The electrostatic surface potential ([77]Fig. 3) indicates
   that alpha 12 may mask some of the charged residues exposed by the two
   helical bundles. In conclusion, the helical domain appears to actually
   consist of two subdomains.

 §5§ [78] Figure 3: Interaction of the C-terminal helix motif alpha 12/13
with the helical and the LG domains. §5§

   [79]Figure 3 : Interaction of the C-terminal helix motif |[alpha]|12/13
   with the helical and the LG domains. Unfortunately we are unable to