James F. Head, Ph.D.

Research

Structural studies of protein-ligand and protein-protein interactions.

Research in our lab focuses on characterizing the molecular details of protein-ligand and protein-protein interactions and the mechanisms by which changes in these interactions regulate physiological processes. These studies have concentrated mainly on the role of the calcium ion in mediating changes in protein-ligand interactions which subsequently may alter protein-protein interactions to modulate function. However, other protein-ligand interactions are also under study including enzyme-effector and protein-protein complexes. X-ray crystallography is a major tool used in these studies.

1. The crystal structure of the calcium-dependent photoprotein aequorin.

This structure shows how the protein binds the ligand coelenterazine creating an environment where the oxygenated intermediate is stabilized. Subsequent binding of calcium to the protein then destabilizes the intermediate, permitting completion of the oxidation reaction which leads to light emission.

Ribbon representation of the secondary structure elements of aequorin, showing the bound coelenterazine peroxide as a stick model.

MarR is a regulator of multiple antibiotic resistance in Escherichia coli.  It is the prototypic member of a family of regulatory proteins found in the Bacteria and the Archae that play important roles in the development of antibiotic resistance, a global health problem.  This is the first reported crystal structure of a member of the MarR protein family.  The structure shows MarR as a dimer with each subunit containing a winged-helix DNA binding motif. In this structure, the protein is bound to an inhibitor, salicylate.

Surface representation of MarR	GRASP surface showing charge distribution

MarR was originally identified as a component of the E. coli marRAB locus and negatively regulates expression of this operon.  Proteins of the MarR family control an assortment of biological functions including resistance to multiple antibiotics, organic solvents, household disinfectants, and oxidative stress agents, collectively termed the multiple antibiotic resistance (Mar) phenotype.  These proteins also regulate the synthesis of pathogenic factors in microbes that infect humans and plants.  Inactivation of MexR, a family member in P. aeruginosa, leads to the overexpression of a multidrug efflux system, which is a major determinant for the very broad resistance phenotype observed in this host.

Secondary structure elements of MarR are shown color-coded to correspond to the ribbon diagram above.
In the sequences, identity is indicated by red, high conservation by yellow, lower conservation by blue.

+ indicates residue involved in the hydrophobic core of the N-C terminal domain * indicates residues involved in the hydrophobic core of the DNA binding domain

The Mar phenotype in E. coli is attributed largely to the action of MarA, the expression of which is regulated by MarR.  MarA is a transcription factor that autoactivates expression of the marRAB operon and regulates the expression of a global network of more than 60 chromosomal genes. Constitutive overexpression of MarA or a MarA homolog in many of these strains is a key contributor to the maintenance of the resistance phenotype, particularly with respect to the fluoroquinolones, and recent studies have documented the selection of Mar mutants, bearing mutations in MarR, MexR, or other homologous loci, in E. coli, Pseudomonas aeruginosa, and other organisms during antimicrobial chemotherapy.  These data strongly support a role for MarR as a critical “stepping stone” toward the failure of antimicrobial chemotherapy.

We are pursuing further studies on MarR and it's interactions.

3. Structure of the actin:vitamin D-binding protein

Vitamin D-binding protein (DBP), also known as group specific component (Gc), is a polymorphic serum glycoprotein with multiple functions that include vitamin D sterol-transport and prevention of arterial congestion in the events of cell-injury and lysis by binding actin released into plasma.  In addition, DBP binds saturated and unsaturated fatty acids with moderate affinity (Ka=10^5-6M^-1) and chemotactic agents.  DBP is also detected on the surface of lymphocytes, neutrophils and monocytes from blood.  The physiological implications of these cell-associative properties are currently uncertain.

DBP binds vitamin D3 and all its major metabolites with high affinity (Ka=10^7-10M^-1).  In plasma, DBP binds vitamin D3 and transports this seco-steroid to the liver to form 25-hydroxyvitamin D3 (25-OH-D3); the latter in turn is transported (by DBP) to kidney to form 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Finally, 1,25(OH)2D3, the active form of vitamin D hormone, is delivered to target tissues by DBP.  Thus biological functions of 1,25(OH)2D3, e.g. calcium and phosphorus homeostasis, immune-modulation, regulation of growth and maturity of normal and malignant cells, are intimately dependent on DBP.

DBP also plays an integral role in a circulating actin-scavenger system in plasma that removes actin released into the circulation following cell damage. In this system, it is believed that  plasma gelsolin severs filaments of free actin (F-actin), DBP binds monomeric actin (G-actin) with high affinity, Ka=10⁸M^-1 , and the DBP-actin complex is rapidly cleared from the circulation, preventing the harmful effects of polymeric actin clogging arteries.  There is significant evidence for this clearance process in a variety of disease states.  DBP-actin complex is found in the serum of man and animals sustaining injuries/inflammation e.g. trophoblastic emboli, severe actute hepatitis, acute lung injury etc.

Actin - Orange subdomain 1, Green subdomain 2, Yellow subdomain 3, Red subdomain 4
DBP - Blue domain 1, Magenta domain 2, Cyan domain 3 - (color-coded as in reference 29).
ATP and Ca2+ (green) are shown as space-filling representations.

The crystal structure of the actin:DBP complex shows one molecule of each protein bound together by extensive ionic, polar and hydrophobic interactions. It includes an ATP and a calcium ion bound to actin, but no evidence of vitamin D metabolites bound to the DBP. Both actin and DBP are multidomain molecules, two major domains in actin and three in DBP. All of these domains contribute to the interaction between the molecules. DBP enfolds the end of the actin molecule, principally in actin subdomain 3 but with additional interactions in actin subdomain 1. This orientation is similar to the binding of profilin to actin, as predicted from previous studies. The more extensive interactions of DBP gives an affinity for actin some three orders of magnitude higher than profilin. The larger “footprint” of DBP on actin also leads to an overlap with the actin-binding site of gelsolin domain I.

Molecules are aligned based on least square superimposition of the actin in each structure, although only the actin of the DBP-actin complex is shown.

Red - actin from DBP-actin complex, blue - DBP from DBP-actin complex, cyan – Gelsolin domain 1 (Protein Databank Code 1EQY), magenta – Profilin (Protein Databank Code 2BTF), green-DNAse (Protein Databank Code 1ATN)

 4. Structure of surfactant proteins A and D

Four major proteins are found in the fluid lining the lung surface, these have been designated surfactant proteins A-D. Two of these, surfactant proteins A and D (SP-A and SP-D), have important roles in innate immunity, providing strategically-placed, key components in our defense against inhaled pathogens.

SP-A and SP-D are homologous proteins belonging to the collectin family of  lectins. Despite many common sequence and structural features, the two proteins have distinct molecular targets. We are working to provide a more detailed structural characterization of the proteins and their interactions to better understand the mechanisms employed by these proteins in targeting surface molecular features on pathogens.

SP-A and SP-D form homotrimers in which the ligand-binding function is provided by a headpiece, carbohydrate recognition (CRD), domain on each subunit. These CRDs form trimers through neck domains and collagen-like tail domains. The tail domain contributes to higher level oligomerization seen in both proteins and the head and neck together (NCRD) form the minimal trimer unit able to bind to target ligands. It is these NCRDs that have been the main focus of our studies.

We have determined the first crystal structure of the SP-A NCRD and evaluated its relationship to the SP-D NCRD structure.

Ribbon diagram of SP-A NCRD subunit and sequence alignment of some other collectins NCRDs.

Superimpostion of ligand binding to SP-D, green-maltose, magenta-maltotriose

Selected Publications:

The crystal structure of the photoprotein aequorin at 2.3A resolution. (2000) Head, J.F. Inouye, S., Teranishi, K. and Shimomura, O. Nature, 405, 372-376. 

A crystal structure of the nucleoplasmin-core decamer suggests a model for histone octamer and nucleosome assembly. (2001.)  S. Dutta, I.V. Akey, C. Dingwall, K.L. Hartman, T. Laue, R.T. Nolte, J.F. Head, C.W. Akey. Molecular Cell 8: 841-853. 

The crystal structure of MarR a regulator of multiple antibiotic resistance at 2.3 Å resolution. (2001) M. N. Alekshun, S. B. Levy, T. R. Mealy, B. A. Seaton and J. F. Head. Nature Structural Biology  8, 710-714. 

J.F. Head , N. Swamy, R. Ray Crystal Structure of the Complex between Actin and Human Vitamin D-Binding Protein at 2.5 A Resolution. Biochemistry. (2002) Jul 23;41(29):9015-20. 

Head JF, Mealy TR, McCormack FX, Seaton BA (2003) Crystal structure of trimeric carbohydrate recognition and neck domains of surfactant protein A. J. Biol. Chem. 278:43254-43260. 

Namboodiri VM, Akey IV, Schmidt-Zachmann MS, Head JF, Akey CW. (2004) The Structure and Function of Xenopus NO38-Core, a Histone Chaperone in the Nucleolus. Structure  12:2149-60. 

Meng, J., Vardar, D., Wang, Y., Guo, H-W., Head, J.F. and McKnight, CJ. (2005) The High Resolution Crystal Structures of Villin Headpiece and Mutants with Reduced F-Actin Binding Activity. Biochemistry 44:11963-73. 

Erika C Crouch, Kelly Smith, Barbara McDonald, David Briner, Bruce Linders, Joseph McDonald, Uffe Holmskov, James Head, and Kevan Hartshorn. (2006) Species Differences in the Carbohydrate Binding Preferences of Surfactant  Protein D.
Prepub: http://ajrcmb.atsjournals.org/cgi/content/abstract/35/1/84 

Erika Crouch, Barbara McDonald, Kelly Smith, Tanya Cafarella, Barbara Seaton, and James Head. (2006) Contributions of phenylalanine 335 to ligand recognition by human surfactant protein D: Ring interactions with SP-D ligands.
Prepub: http://www.jbc.org/cgi/content/abstract/281/26/18008
 

Teaching:

Contact Us

Boston University School of Medicine
Department of Physiology and Biophysics
715 Albany St Boston MA 02118-2526

Phone:(617) 638-4396
Fax: (617) 638-4273
e-mail: head@bu.edu

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