Research

We do this project in collaboration with Walter Atwood (Brown University), Robert Garcea (University of Colorado, Boulder), Dale Mierke (Dartmouth College), the Consortium for Functional Glycomics Core H (Emory University) and Ten Feizi (Imperial College). It is funded by the German Research Foundation (SFB 685) and an NIH program project grant with Drs. Atwood and Mierke.

Structure and function of enzymes involved in the bacterial cell wall synthesis and degradation

PknB. Staphylococcus aureus (S. aureus) is a gram-positive bacterium colonising mainly the upper respiratory tract and the skin. The pathogen causes disease patterns ranging from mild skin or respiratory infections to life-threatening diseases such as endocarditis, sepsis or toxic shock syndrome. These life-threatening infections occur mostly in persons with multiple risk factors for infections. The increasing amount of multi-drug resistant strains has become a severe problem in the treatment of these bacterial infections. Therefore, new approaches for therapy are urgently needed due to mounting resistance against the existing drugs that target S. aureus. Protein kinases are important enzymes for regulation of processes and signal transduction pathways in prokaryotes and eukaryotes. While many microorganisms encode several serine/threonine protein kinases (STPKs), S. aureus encodes only one such protein, PknB. Thus, PknB provides a promising target in the treatment of S. aureus infections. We therefore attempt to solve the X-ray crystallographic structure of PknB at high resolution.

MprF. Defensins are positively charged antimicrobial peptides of the innate immune system that protect humans against microbial infections. MprF is an integral membrane protein rendering S. aureus insensitive against these defensins. A synthethase activity in MprF adds l-lysin to phosphatidylglycerol. The resulting l-lysin-phosphatidylglycerol is built into the bacterial membrane. This leads to a change in the net surface charge of the bacterium, which in turn prevents the cationic defensins from attacking the bacterium. This makes MprF a possible drug target to defeat S. aureus. We are working on the purification of the MprF synthetase domain in order to crystallize it and solve its X-ray structure.

Amidase. The major autolysins (Atl) of S. epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. We attempt to solve the resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. We are also trying to identify the minimal peptidoglycan fragment that can be used as a substrate by the enzyme using molecular docking and digestion assays. The results will provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.

S. aureus is a gram-positive bacterium that colonizes the anterior nares of at least one third of the human population. It is also known to cause a variety of infections, ranging from superficial lesions to severe systemic infections. The pathogenicity of this organism largely depends on the successful adaptation to the human host and the environmentally coordinated expression of virulence factors. A network of interacting regulons tightly controls the virulence factor expression pattern during the growth cycle of S. aureus.

The agr (accessory gene regulator), the SarA (staphylococcal accessory regulator), the Sigma (alternative sigma factor) and the Rot (regulator of toxins) regulons are among the best- characterized virulence-associated regulons. More recently, the sae locus was described as a regulatory locus consisting of four open reading frames, two of which encode the response regulator SaeR and the sensor histidin kinase SaeS, respectively. The sae operon gives rise to the two-component system SaeRS, which is now known as another crucial regulator of virulence gene expression in S. aureus. Expression of a multitude of virulence factors, such as serine protease SspA, thermonuclease Nuc, coagulase Coa, alpha-hemolysin Hla, beta-hemolysin Hlb and fibronectin binding protein FnbA, was shown to be activated by the SaeRS system. It has also been proposed that SaeRS regulates the synthesis of extracellular proteins downstream of the agr regulon and might modify quorum sensing-dependent regulation. More recently, proteomic and transcriptomic studies were carried out in the S. aureus, yielding a the complete set of proteins that are most likely under the control of SaeRS. Most of these proteins play a role in immune evasion, while others are especially involved in adhesion to host cells. Although the detailed functionality of these virulence factors is not known to date, we will assign a function by structural genomics. Therefore our research is designed to determine the structure of these proteins and then to deduce a biological function by comparison to structurally homologous proteins. This work is funded by SFB/Transregio 34.

Adenylyl cyclase from Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the most effective pathogens. It claims about 2 million lives each year and lurks in one-third of the world’s population. It is possible that M. tuberculosis has evolved eukaryotic-like signal transduction mechanisms which may be capable of modulating host cell signal pathways. In M. tuberculosis, 15 genes encode class III adenylyl cyclases of variant domain compositions, suggesting that cAMP formation may contribute to the pathogenesis.

We are interested in the structure, function and regulation of the mammalian-like adenylyl cyclase Rv1625c from M. tuberculosis. Adenylyl cyclase catalyzes the formation of cAMP which is a key signaling molecule in virtually all living organisms. Up to now, the most intensively studied adenylyl cyclases are nine mammalian membrane-bound enzymes. They contain two putative membrane domains; each with six transmembrane spans, and two complementary catalytic domains. The structure of the catalytic domains is well characterized; they form a pseudoheterodimer. The membrane domains account for about 40% of the protein, the function of which is unknown. Hitherto, no structure details are available for the holoenzymes because of the scarcity of the proteins.

Rv1625c from M. tuberculosis is a mammalian-like adenylyl cyclase with six putative transmembrane helices and a catalytic domain, corresponding exactly to one half of the nine mammalian membrane-bound adenylyl cyclases. It is active as homodimer. Therefore, the structure determination of Rv1625c is important and urgent for exploring the structure, the function and the regulation of the membrane-bound adenylyl cyclases, and for answering the questions about the involvement of adenylyl cyclases in the pathogenesis of M. tuberculosis.

Stuctural analysis of the NKC encoded, C-type lectin like receptor-ligand pair Nkp80 and AICL

Natural killer cells (NK cells) are lymphocytes of the innate immune system that play an important role in immune responses against certain pathogen-infected cells and tumors. Activity of NK cells is tightly regulated by the balance of activating and inhibitory signals delivered through a diverse set of cell surface receptors. Most of the NK receptors are members of the immunglobulin superfamily (IgSF) or the C-type lectin superfamily (CLSF) with the latter ones mostly being homo- or heterodimers of type II transmembrane proteins containing a single extracellular C-type lectin-like domain (CTLD). The CTLD was originally identified in certain lectins where it serves as a Ca²⁺-dependent carbohydrate recognition domain. However, most NKC-encoded CTLR have lost critical CTLD residues for carbohydrate binding and interact with protein ligands instead.

Although the CTLD-fold is a very common motive among proteins involved in the innate immune response, CTLDs from different proteins show only little sequence similarity (~20% to 40%) and have no conserved binding surface for dimerization or their ligand binding epitopes underlining the need for continued structure determination, even within a family of homologous protein modules. The crosstalk between the NK-cell receptor NKp80 and its ligand, the activation induced C-type lectin (AICL), may play a crucial role in immunosurveillance of viral and bacterial infections, malignancy, and the pathogenesis of autoimmune diseases, but the signal-transducing elements associated with NKp80 or AICL have not yet been defined. In addition, it is still unclear, whether NKp80 has more than one ligand or functions as coreceptor, e.g., with NKp46. Furthermore, only very little is known about AICL and its molecular binding properties to NKp80. Therefore, knowledge about the 3D-structure of both AICL and NKp80 as well as of their complex may help to get answers to the many questions left in the contect of NKp80-mediated NK-cells response and the role of its newly discovered ligand AICL.

Deciphering the structure of Insulin Receptor Substrate

Insulin Receptor Substrate-1 (IRS-1) is a key element in Insulin and Insulin-like Growth Factor (IGF) actions. It transduces pleiotropic effects on cellular functions and modulates the processes such as metabolism, growth, cell differentiation and survival. Human IRS-1 is a ~170 KDa phospho-protein and is composed of 1242 amino acids. It contains Pleckstrin-Homology (PH) and Phospho-tyrosine binding (PTB) domains at the N-terminus that are involved in its targeting and binding to receptor tyrosine kinase(s). The remaining part of IRS1 (~980 amino acids) are collectively referred to as the "C-terminal tail" of IRS-1. The "C-terminal tail" region has various physiologically relevant motifs that are the targets of multiple kinases and other regulatory proteins. We propose that the "C-terminal tail" is a multi-domain region that contains numerous functional domains for protein-protein interaction and intracellular signal transduction. The aim of this project is to elucidate the structure of the "C-terminal tail" region of the IRS-1. This will ultimately help us to highlight the structural basis of the IRS-1 function.