Indian Journal of Biochemistry & Biophysics Vol. 40, February 2003, pp. 14-21 Crystal structure of human seminal diferric lactoferrin at 3.4 Å resolution Janesh Kumar 1 , Wolfgang Weber 2 , Sabine Münchau 2 , Savita Yadav 1 , S Bhaskar Singh 1 , K Saravanan 1 , M Paramasivam 1 , Sujata Sharma 1 , Punit Kaur 1 , A Bhushan 1 , A Srinivasan 1 , Christian Betzel 3 and T P Singh 1 * 1 Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110029, India 2 Universitätsklinikum Hamburg-Eppendorf, IMBM, Martinistrasse 52, D-20246 Hamburg, Germany 3 Institute of Medical Biochemistry and Molecular Biology, C/o DESY, Geb 22a Notkestrasse 85, 22603, Hamburg, Germany Received 29 October 2002; revised 16 January 2003 Lactoferrin was purified from human seminal fluid obtained from the semen bank. The purified samples were saturated with Fe 3+ and crystallized by microdialysis method. The crystals belong to orthorhombic space group P2 1 2 1 2 1 with a = 55.9 Å, b = 97.2 Å, c = 156.1 Å and Z = 4. The structure was determined with molecular replacement method and refined to an R factor of 18.7% for all the data to 3.4 Å resolution. The overall structure of seminal lactoferrin is similar to human colostrum lactoferrin. The amino acid sequence of seminal lactoferrin shows that it has one amino acid less than human colostrum lactoferrin and the structure of its N-terminal region is far more ordered than other lactoferrins. The structure of the iron- binding site and its immediate surroundings indicate well defined features. Lactoferrin an 80 kDa, single-chain, iron-binding glycoprotein consists of two similar sized homologous N and C lobes, which are further divided into two similar sized domains: N1 and N2 in the N-lobe and C1 and C2 in the C-lobe. The iron-binding sites are located within the interdomain cleft of each lobe. It has the ability to bind, very tightly but reversibly, two Fe 3+ ions, together with two CO 3 2− ions. The synergistic relationship between metal binding and anion binding is one of its unique features not observed in other metal binding proteins 1,2,3 Crystal structures of diferric forms of human 4,5 , bovine 6 , equine 7 and buffalo 8,9 lactoferrins reveal that the two domains are closed over an Fe 3+ ion. For the iron-free apo forms, X-ray crystallographic studies on human 10 , equine 11,12 and camel 13 lactoferrins and solution scattering analysis 14 have shown that the apolactoferrins assume conformations with variable domain arrangements. The intermediates in the iron- binding pathway using camel apolactoferrin as a starting point 3 and iron-release pathway using goat diferric lactoferrin as a starting point 15 have revealed the existence of iron-bound tetrahedral open and closed forms separately. Although, the lactoferrins from various species share many structural and functional features, they differ in several others 16 . The behaviour of iron- binding and iron-release against pH shows remarkable variations. The numbers of carbohydrate attachments and their locations differ greatly 17 . The processes of domain-closing and domain-opening show remarkable differences. There are observed variations in functions other than the iron-binding and iron- release. So far, the lactoferrin structures from six species 4,6,7,8,13,15 have been determined, but all of them have used colostrum as the source. The primary consideration for choosing colostrum has been due to high concentrations of lactoferrin in it. However, there are a number of other secretary sources of lactoferrin such as tears, saliva, sweat, genital secretions, synovial fluid etc, but no structural report is available to highlight the similarities and differences between lactoferrins from different secretions. In order to understand the role of lactoferrin in human reproductive physiology and to evaluate the tissue related structural and functional variations, we have isolated lactoferrin from human seminal fluid and have determined its detailed three- dimensional structure. The results of structure analysis of seminal human lactoferrin (sLf) are presented here. Materials and Methods Isolation and purification Samples of human semen obtained from a laboratory specialized in fertility analyses __________ *For correspondence E-mail: tps@aiims.aiims.ac.in Tel: +91-11-653 3931 Fax: +91-11-686 2663