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The Structural Biology Research group aims to characterise the molecular mechanisms by which molecules of the innate immune system select, recognise and bind to their natural targets and effect clearance through interaction with components of immune system pathways. This structural immunology includes ligand/pathogen recognition by the pentraxins CRP and SAP from human and a variety of other sources including Limulus, rat and fish, and by collectins including hSP-D, CL46 and conglutinin. In addition to crystallography, our structural investigations utilise complementary techniques including SRCD and cryoEM.

Lung surfactant protein D (SP-D) can directly interact with carbohydrate residues on pulmonary pathogens and allergens, stimulate immune cells, and manipulate cytokine and chemokine profiles during the immune response in lungs. The high resolution crystal structures, both native and ligand bound, of a therapeutically active recombinant fragment of SP-D define the fine detail of the mode and nature of carbohydrate recognition and provide insights into how a small fragment of human SP-D can bind to allergens/antigens or whole pathogens, and at the same time recruit and engage effector cells and molecules of humoral immunity.

References: Shrive et al. (2003) Journal of Molecular Biology. 331, 509-523.
Kishore et al. (2006) Molecular Immunology, 43, 1293-1315.
Shrive et al. (2009) Journal of Molecular Biology. 394, 776-788.

Image - T J Greenhough & A K Shrive © Keele University
Human C-reactive protein is a trace plasma protein which exhibits rapid increases in concentration of up to 1000-fold in response to tissue damage and inflammation. As the classical acute-phase reactant, CRP is used almost universally as a clinical indicator of inflammation and underlying infection.
The structure contains a remarkable crystal contact, where the calcium binding loop including Glu147 from one protomer coordinates into the calcium site of a protomer in a symmetry related pentamer. The Glu147-Phe146 dipeptide from this loosely associated 140-150 loop mimics phosphate-choline (PC) binding in the accepting protomer. The movement of the loop also results in the loss of calcium in the donating protomer where large concerted movements of the structure, involving 43-48, 67-72 and 85-91 are seen. A striking structural cleft on the pentameric face opposite to the PC binding site, suggests an important functional role, perhaps in complement activation (see below). Neither binding of simple ligands nor calcium depletion affects the site.

References: Shrive et al. (1996) Nature Struct. Biol. 3, 346-354.
Ramadan et al. (2002) Acta Cryst. D58, 992-1001.

The host defence functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The 3-dimensional structure of human CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. The shallow end of the pocket is bounded by the 112-114 loop, residues 86-92 (the inner loop), the C-terminus of the protomer, and the C-terminus of the pentraxin alpha-helix 169-176. Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp112 and Tyr175 are important contact residues for C1q binding, that Glu88 influences the conformational change in C1q necessary for complement activation, and that Asn158 and His38 probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP.

Reference: Agrawal et al. (2001) J. Immunol. 166, 3998-4004.

The serum-amyloid-P-component-like pentraxin from Limulus polyphemus is a recently discovered pentraxin species and important effector protein of the hemolymph immune system. Our results show that, contrary to popular opinion, both CRP and SAP are present in Limulus haemolymph. The refined three-dimensional structures determined by X-ray crystallography (the first invertebrate pentraxin structures) reveal two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. Each aggregate is constructed from a similar dimer of protomers which is repeated to make up the ring structure. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket.

References: Shrive et al. (1999) J. Mol. Biol. 290, 997-1008.
Tharia et al. (2002) J. Mol. Biol. 316, 583-597.
Shrive et al. (2009) J. Mol. Biol. 386, 1240-1254.

Structural Biology Research Highlights
The Structural Biology Research group aims to characterise the molecular mechanisms by which molecules of the innate immune system select, recognise and bind to their natural targets and effect clearance through interaction with components of immune system pathways. This structural immunology includes ligand/pathogen recognition by the pentraxins CRP and SAP from human and a variety of other sources including Limulus, rat and fish, and by collectins including hSP-D, CL46 and conglutinin. In addition to crystallography, our structural investigations utilise complementary techniques including SRCD and cryoEM.
Trevor Greenhough Dr. Annette Shrive
High resolution structures of native and ligand bound surfactant protein D
Lung surfactant protein D (SP-D) can directly interact with carbohydrate residues on pulmonary pathogens and allergens, stimulate immune cells, and manipulate cytokine and chemokine profiles during the immune response in lungs. The high resolution crystal structures, both native and ligand bound, of a therapeutically active recombinant fragment of SP-D define the fine detail of the mode and nature of carbohydrate recognition and provide insights into how a small fragment of human SP-D can bind to allergens/antigens or whole pathogens, and at the same time recruit and engage effector cells and molecules of humoral immunity. References: Shrive et al. (2003) Journal of Molecular Biology. 331, 509-523. Kishore et al. (2006) Molecular Immunology, 43, 1293-1315. Shrive et al. (2009) Journal of Molecular Biology. 394, 776-788.
Structure of Human C-reactive protein
Image - T J Greenhough & A K Shrive © Keele University Human C-reactive protein is a trace plasma protein which exhibits rapid increases in concentration of up to 1000-fold in response to tissue damage and inflammation. As the classical acute-phase reactant, CRP is used almost universally as a clinical indicator of inflammation and underlying infection. The structure contains a remarkable crystal contact, where the calcium binding loop including Glu147 from one protomer coordinates into the calcium site of a protomer in a symmetry related pentamer. The Glu147-Phe146 dipeptide from this loosely associated 140-150 loop mimics phosphate-choline (PC) binding in the accepting protomer. The movement of the loop also results in the loss of calcium in the donating protomer where large concerted movements of the structure, involving 43-48, 67-72 and 85-91 are seen. A striking structural cleft on the pentameric face opposite to the PC binding site, suggests an important functional role, perhaps in complement activation (see below). Neither binding of simple ligands nor calcium depletion affects the site. References: Shrive et al. (1996) Nature Struct. Biol. 3, 346-354. Ramadan et al. (2002) Acta Cryst. D58, 992-1001.
Topology of the C1q-binding site on human CRP
The host defence functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The 3-dimensional structure of human CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. The shallow end of the pocket is bounded by the 112-114 loop, residues 86-92 (the inner loop), the C-terminus of the protomer, and the C-terminus of the pentraxin alpha-helix 169-176. Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp112 and Tyr175 are important contact residues for C1q binding, that Glu88 influences the conformational change in C1q necessary for complement activation, and that Asn158 and His38 probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP. Reference: Agrawal et al. (2001) J. Immunol. 166, 3998-4004.

Structures of Limulus SAP
The serum-amyloid-P-component-like pentraxin from Limulus polyphemus is a recently discovered pentraxin species and important effector protein of the hemolymph immune system. Our results show that, contrary to popular opinion, both CRP and SAP are present in Limulus haemolymph. The refined three-dimensional structures determined by X-ray crystallography (the first invertebrate pentraxin structures) reveal two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. Each aggregate is constructed from a similar dimer of protomers which is repeated to make up the ring structure. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. References: Shrive et al. (1999) J. Mol. Biol. 290, 997-1008. Tharia et al. (2002) J. Mol. Biol. 316, 583-597. Shrive et al. (2009) J. Mol. Biol. 386, 1240-1254.
	This page was last updated on June 25, 2010

Structural Biology Research Highlights

Trevor Greenhough Dr. Annette Shrive

High resolution structures of native and ligand bound surfactant protein D

Structure of Human C-reactive protein

Topology of the C1q-binding site on human CRP

Structures of Limulus SAP

Trevor Greenhough
Dr. Annette Shrive