Rolf Hilgenfeld

The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor

A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiological agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, we report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to our knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.

Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain.

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, Mpro, a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus Mpro is reported. The structure was refined to 1.96 Å resolution and revealed three dimers in the asymmetric unit. The mutual arrangement of the protomers in each of the dimers suggests that Mpro self‐processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin‐like fold that is connected by a 16 residue loop to an extra domain featuring a novel α‐helical fold. Molecular modelling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of Mpro substrates. Interactions involving the N‐terminus and the α‐helical domain stabilize the loop in the orientation required for trans‐cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.

Utility of homology models in the drug discovery process

Abstract Advances in bioinformatics and protein modeling algorithms, in addition to the enormous increase in experimental protein structure information, have aided in the generation of databases that comprise homology models of a significant portion of known genomic protein sequences. Currently, 3D structure information can be generated for up to 56% of all known proteins. However, there is considerable controversy concerning the real value of homology models for drug design. This review provides an overview of the latest developments in this area and includes selected examples of successful applications of the homology modeling technique to pharmaceutically relevant questions. In addition, the strengths and limitations of the application of homology models during all phases of the drug discovery process are discussed.

Conformational Antagonism between Opposing Active Sites in a Bifunctional RelA/SpoT Homolog Modulates (p)ppGpp Metabolism during the Stringent Response

Abstract Enzymes of the Rel/Spo family enable bacteria to survive prolonged periods of nutrient limitation by producing an intracellular signaling alarmone, (p)ppGpp, which triggers the so-called stringent response. Both the synthesis of (p)ppGpp from ATP and GDP(GTP), and its hydrolysis to GDP(GTP) and pyrophosphate, are catalyzed by Rel/Spo proteins. The 2.1 A crystal structure of the bifunctional catalytic fragment of the Rel/Spo homolog from Streptococcus dysgalactiae subsp. equisimilis , Rel Seq , reveals two conformations of the enzyme corresponding to known reciprocal activity states: (p)ppGpp-hydrolase-OFF/(p)ppGpp-synthetase-ON and hydrolase-ON/synthetase-OFF. The hydrolase and synthetase domains bear remarkable similarities to the catalytic domains of the cyclic phosphodiesterase and nucleotidyltransferase superfamilies, respectively. The active sites, separated by more than 30 A, contain bound nucleotides including an unusual (p)ppGpp derivative, GDP-2′:3′-cyclic monophosphate. Reciprocal regulation of the antagonistic catalytic activities, suggested by the structure, is supported by mutagenesis experiments and appears to involve ligand-induced signal transmission between the two active sites.

More hydrogen bonds for the (structural) biologist

Why does a given protein structure form and why is this structure stable? These fundamental biochemical questions remain fascinating and challenging problems because the physical bases of the forces that govern protein structure, stability and folding are still not well understood. Now, a general concept of hydrogen bonding in proteins is emerging. This concept involves not only N-H and O-H donor groups, but also C-H, and not only N and O as acceptor groups, but also pi-systems. We postulate that the incorporation of the entirety of these interactions leads to a more complete description of the problem, and that this could provide new perspectives and possibly new answers.

Structure and functionality in flavivirus NS-proteins: perspectives for drug design.

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer

Membrane‐bound glutamate carboxypeptidase II (GCPII) is a zinc metalloenzyme that catalyzes the hydrolysis of the neurotransmitter N‐acetyl‐L‐aspartyl‐L‐glutamate (NAAG) to N‐acetyl‐L‐aspartate and L‐glutamate (which is itself a neurotransmitter). Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain. Here, we report crystal structures of the extracellular part of GCPII in complex with both potent and weak inhibitors and with glutamate, the product of the enzymes hydrolysis reaction, at 2.0, 2.4, and 2.2 Å resolution, respectively. GCPII folds into three domains: protease‐like, apical, and C‐terminal. All three participate in substrate binding, with two of them directly involved in C‐terminal glutamate recognition. One of the carbohydrate moieties of the enzyme is essential for homodimer formation of GCPII. The three‐dimensional structures presented here reveal an induced‐fit substrate‐binding mode of this key enzyme and provide essential information for the design of GCPII inhibitors useful in the treatment of neuronal diseases and prostate cancer.

An α to β conformational switch in EF-Tu

Abstract Background The bacterial elongation factor EF-Tu recognizes and transports aminoacyl-tRNAs to mRNA-programmed ribosomes. EF-Tu shares many structural and functional properties with other GTPases whose conformations are regulated by guanine nucleotides. Results An intact form of Escherichia coli EF-Tu complexed with GDP has been crystallized in the presence of the EF-Tu-specific antibiotic GE2270 A. The three-dimensional structure has been solved by X-ray diffraction analysis and refined to a final crystallographic R factor of 17.2% at a resolution of 2.5 a. The location of the GE2270 A antibiotic-binding site could not be identified. Conclusions The structure of EF-Tu–GDP is nearly identical to that of a trypsin-modified form of EF-Tu–GDP, demonstrating conclusively that the protease treatment had not altered any essential structural features. The present structure represents the first view of an ordered Switch I region in EF-Tu–GDP and reveals similarities with two other GTPases complexed with GDP: Ran and ADP-ribosylation factor-1. A comparison of the Switch I regions of the GTP and GDP forms of EF-Tu also reveals that a segment, six amino acids in length, completely converts from an α helix in the GTP complex to β secondary structure in the GDP form. The α to β switch in EF-Tu may represent a prototypical activation mechanism for other protein families.

Two non-proline cis peptide bonds may be important for factor XIII function.

The structure of recombinant human cellular factor XIII zymogen was solved in its monoclinic crystal form and refined to an R‐factor of 18.3% (R free=23.6%) for all data between 40.0 and 2.1 Å resolution. Two non‐proline cis peptide bonds were detected. One is between Arg310 and Tyr311 close to the active site cysteine residue (Cys314) and the other is between Gln425 and Phe426 at the dimerization interface. The structure and the role of these cis peptides are discussed in the light of their possible importance for factor XIII function.

The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.