Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major

Abstract

The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (Rfree = 0.229) at 1.60 A resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif.

Figure 1

Truncation variants of target ORF Lmaj006129. Structures were determined for the truncation variant Lmaj006129AAH and for the full-length target Lmaj006129AAA.

Figure 2

Multiple sequence alignment of PF04543 family members. TrEMBL sequence identifiers and residues extents are shown on the right. Secondary-structure elements and residue numbering are from the present L. major structure (PDB code 2ar1). Sequence alignment is based on CLUSTALW analysis of 106 sequence family members. Residue positions are colored if the hydropathy class is conserved in  >50% of the family members. Below this is shown the sequence fingerprint of P. horikoshii protein PH1033 (PDB code 1wmm) from Pfam sequence family PF01878, aligned based on structural similarity to the L. major structure. Residues lining a binding pocket observed in the four sequences with known structure (PDB codes at far right) are indicated by brown dots. This figure was generated using T_EXshade (Beitz, 2000 ▶).

Figure 3

(a) A ribbon diagram showing the tertiary structure of Lmaj006129AAA color coded from the amino-terminus (blue) to the carboxy-terminus (red) with secondary-structure elements labeled as β1 (13–18), α1 (24–32), β2 (33–35), α2 (41–51), β3 (56–61), β4 (68–81), α3 (83–87), β5 (105–120), α4 (121–125), α5 (126–131), β6 (143–146), 6 (147–162). (b) Stereo figure depicted as a coil with every tenth residue numbered. It is in the same orientation and color scheme as (a).