The 1.7 A Ê Crystal Structure of BPI: A Study of How Two Dissimilar Amino Acid Sequences can Adopt the Same Fold Gary Kleiger 1 , Lesa J. Beamer 2 , Robert Grothe 1 , Parag Mallick 1 and David Eisenberg 1 * 1 UCLA-DOE Laboratory of Structural Biology and Molecular Medicine, Molecular Biology Institute, UCLA BOX 951570, Los Angeles CA 90095-1570, USA 2 Biochemistry Department University of Missouri- Columbia, Columbia MO 65211, USA We have extended the resolution of the crystal structure of human bac- tericidal/permeability-increasing protein (BPI) to 1.7 A Ê . BPI has two domains with the same fold, but with little sequence similarity. To under- stand the similarity in structure of the two domains, we compare the corresponding residue positions in the two domains by the method of 3D-1D pro®les. A 3D-1D pro®le is a string formed by assigning each pos- ition in the 3D structure to one of 18 environment classes. The environ- ment classes are de®ned by the local secondary structure, the area of the residue which is buried from solvent, and the fraction of the area buried by polar atoms. A structural alignment between the two BPI domains was used to compare the 3D-1D environments of structurally equivalent positions. Greater than 31 % of the aligned positions have conserved 3D- 1D environments, but only 13 % have conserved residue identities. Anal- ysis of the 3D-1D environmentally conserved positions helps to identify pairs of residues likely to be important in conserving the fold, regardless of the residue similarity. We ®nd examples of 3D-1D environmentally conserved positions with dissimilar residues which nevertheless play similar structural roles. To generalize our ®ndings, we analyzed four other proteins with similar structures yet dissimilar sequences. Together, these examples show that aligned pairs of dissimilar residues often share similar structural roles, stabilizing dissimilar sequences in the same fold. # 2000 Academic Press Keywords: BPI; X-ray crystallography; 3D-1D environment; domain; fold *Corresponding author Introduction Proteins with sequence similarity also display structural similarity. However, many proteins with no apparent sequence similarity display the same folds. For example, the mitochondrial enzyme rho- danese contains two domains of similar structure, but little sequence similarity (Hol et al., 1983). This phenomenon is not rare. In fact, the database of distant aligned protein structures (DAPS) has over 1000 examples of structurally similar proteins with less than 25 % sequence identity (Rice & Eisenberg, 1997). Examples of both intra and inter-molecular fold similarity in the absence of amino acid simi- larity is given in Table 1. To study how dissimilar protein sequences adopt similar folds, we analyze the structure of the bactericidal/permeability-increasing protein (BPI). BPI has two domains with the same fold but with dissimilar sequences. Both BPI domains are twisted, anti-parallel b-sheet barrels capped by two a-helices. The domain main-chain atoms can be superimposed without signi®cant deformation (3.0 A Ê rmsd over 173 residues). The BPI domain is to date a unique fold. We take advantage of the apparent domain duplication in BPI to ®nd struc- turally conserved positions for the BPI domain. BPI is a mammalian protein located in polymor- phonuclear neutrophils, a cell of the innate immune response that protects the host during microbial infection (Elsbach & Weiss, 1995). BPI speci®cally binds lipopolysaccharides in the outer- membrane of Gram-negative bacteria. Although E-mail address of the corresponding author: david@mbi.ucla.edu Abbreviations used: BPI, bactericidal/permeability- increasing protein; rmsd, root mean square deviation; FWLO, fractional weighted log-odds. doi:10.1006/jmbi.2000.3805 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 299, 1019±1034 0022-2836/00/041019±16 $35.00/0 # 2000 Academic Press