SummaryProtein design provides an ultimate test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. While progress has been made in designing proteins that mimic native proteins structurally1–3, it is more difficult to design functional proteins4–8. In comparison to recent successes in designing non-metalloproteins4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes5,8,11–20, since protein metal binding sites are much more varied than non-metal containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal binding site properties in silico, as many of the parameters for metal binding sites, such as force fields are ill-defined. Therefore, the successful design of a structural and functional metalloprotein will greatly advance the field of protein design and our understanding of enzymes. Here, we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a heme/non-heme FeB center that is remarkably similar to that in the crystal structure. This designed protein also exhibits NOR activity. This is the first designed protein that models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

Background: Coronavirus spike protein N-terminal domains (NTDs) bind sugar or protein receptors. Results: We determined crystal structure of bovine coronavirus NTD and located its sugar-binding site using mutagenesis. Conclusion: Bovine coronavirus NTD shares structural folds and sugar-binding sites with human galectins and has subtle yet functionally important differences from protein-binding NTD of mouse coronavirus. Significance: This study explores origin and evolution of coronavirus NTDs.

Mammalian aminopeptidase N (APN) plays multifunctional roles in many physiological processes, including peptide metabolism, cell motility and adhesion, and coronavirus entry. Here we determined crystal structures of porcine APN at 1.85 Å resolution and its complexes with a peptide substrate and a variety of inhibitors. APN is a cell surface-anchored and seahorse-shaped zinc-aminopeptidase that forms head-to-head dimers. Captured in a catalytically active state, these structures of APN illustrate a detailed catalytic mechanism for its aminopeptidase activity. The active site and peptidebinding channel of APN reside in cavities with wide openings, allowing easy access to peptides. The cavities can potentially open up further to bind the exposed N terminus of proteins. The active site anchors the N-terminal neutral residue of peptides/proteins, and the peptide-binding channel binds the remainder of the peptides/ proteins in a sequence-independent fashion. APN also provides an exposed outer surface for coronavirus binding, without its physiological functions being affected. These structural features enable APN to function ubiquitously in peptide metabolism, interact with other proteins to mediate cell motility and adhesion, and serve as a coronavirus receptor. This study elucidates multifunctional roles of APN and can guide therapeutic efforts to treat APN-related diseases.M ammalian aminopeptidase N (APN) plays pivotal roles in many physiological processes, such as pain sensation, blood pressure regulation, tumor angiogenesis and metastasis, immune cell chemotaxis, sperm motility, cell-cell adhesion, and coronavirus entry (1). Accordingly, APN is a major target for treatment of diseases that are related to the above physiological processes. It is puzzling how APN is able to possess such a wide range of physiological functions, some of which are seemingly unrelated to its aminopeptidase activity. This study determines the atomic structures of mammalian APN and its complexes with a variety of APN-targeting ligands, providing structural basis for the multifunctional roles of APN and for the development of novel therapy strategies to treat APN-related diseases.The M1-family of metalloenzymes consists of a large number of zinc-dependent aminopeptidases containing a zinc-binding HEXXH motif. As the most extensively studied member in this family, mammalian APN (also known as CD13 or alanine aminopeptidase) is widely expressed on cell surfaces of tissues, such as intestinal epithelia and the nervous system (1). APN preferentially cleaves neutral amino acids, most notably alanine, off the N terminus of peptides. The general catalytic mechanism of M1-family metalloenzymes is believed to be similar to that of prototypic zincpeptidase thermolysin, which involves catalytic water attacking scissile peptide bonds (2), but detailed catalytic mechanisms of these enzymes remain elusive. To date, crystal structures are available for several members of the M1-family metalloenzymes (3-8). However, these enzymes are monomeric intracellu...

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe B site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by ∼110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ∼100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe B site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more welldefined system to address important chemical and biological issues.biomimetic models | heme-copper oxidase | metalloprotein | protein design | protein engineering R ational design of proteins that mimic both structure and function of more complex native enzymes has been a long soughtafter goal, as the process is an ultimate test of our knowledge and an excellent means to develop advanced biocatalysts (1-3). Although designed proteins that model the structure of native enzymes have been known for a while (4-10), successful designs of proteins that mimic both the structure and function of native enzymes have been reported only recently (11-16). While being able to design such functional proteins is laudable, the impact of such an achievement would be greater if the designed proteins can be used to address fundamental issues in chemistry and biology that are difficult to tackle by other methods. One primary example is the roles of conserved glutamates and metal ions in bacterial nitric oxide reductase (NOR) (17)(18)(19).NO is critical for all life (20). Bacterial denitrification is a crucial part of the nitrogen cycle in nature that involves a four-step, five-electron reduction of nitrate (NO 3 − ) to dinitrogen (N 2 ) (17, 19). Bacterial NOR is a membrane-bound protein that catalyzes one step of this process, namely, the two-electron reduction of NO to N 2 O (17, 19). With no crystal or solution structure available for bacterial NOR to date, sequence alignments and homology modeling (21, 22) have indicated that NOR is structurally homologous to the largest subunit (subunit I) of hemecopper oxidases (HCOs) (23), enzymes that catalyze reduction of O 2 to water. The active sites of both NOR and HCO contain a proximal histidine-coo...

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans. Since the summer of 2012, a novel coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged from the Middle East and spread to parts of Europe. MERS-CoV infection often leads to acute pneumonia and renal failure, and the human fatality rate is more than 50% (1, 2). To date, MERS-CoV has infected at least 77 people and was able to be transmitted from human to human. The genomic sequence of MERS-CoV is closely related to the sequences of certain bat coronaviruses (3-5), raising concerns over persistent bat-to-human cross-species transmission of the virus. The clinical signs and epidemic patterns of MERS-CoV are reminiscent of the severe acute respiratory syndrome coronavirus (SARS-CoV), the etiological agent of the worldwide SARS epidemic in 2002-2003 that infected more than 8,000 people with a ϳ10% fatality rate (6, 7). MERS-CoV poses a significant threat to global health and economy.Coronaviruses are enveloped and positive-stranded RNA viruses and can be divided into three major genera, ␣, ␤, and ␥ (8). They mainly cause respiratory, gastrointestinal, and central nervous system diseases in mammals and birds. Coronaviruses recognize a variety of host receptors. Human NL63 respiratory coronavirus (HCoV-NL63) from ␣-genus and SARS-CoV from ␤-genus both recognize angiotensin-converting enzyme 2 (ACE2) as their host receptor (9, 10). Porcine respiratory coronavirus (PRCV) and some other coronaviruses from ␣-genus recognize aminopeptidase N (APN) (11, 12). Mouse hepatitis coronavirus (MHV) from ␤-genus recognizes carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) (13, 14), although certain MHV strains also recognize heparan sulfate (15, 16). Some coronaviruses from each of the three genera recognize sugars (17-20). MERS-CoV belongs to the ␤-genus and uses human dipeptidyl peptidase 4 (DPP4) as its host receptor (21). Receptor recognition is a major determinant of coronavirus host range and tropism.An envelope-anchored trimeric spike p...

Two studies are presented which examined the temporal dynamics of the social-attentive behaviors that co-occur with referent identification during natural parent-child interactions in the home. Study 1 focused on 6.2 hours of videos of 56 parents interacting during everyday activities with their 14–18 month-olds, during which parents uttered common nouns as parts of spontaneously occurring utterances. Trained coders recorded, on a second-by-second basis, parent and child attentional behaviors relevant to reference in the period (40 sec.) immediately surrounding parental naming. The referential transparency of each interaction was independently assessed by having naïve adult participants guess what word the parent had uttered in these video segments, but with the audio turned off, forcing them to use only non-linguistic evidence available in the ongoing stream of events. We found a great deal of ambiguity in the input along with a few potent moments of word-referent transparency; these transparent moments have a particular temporal signature with respect to parent and child attentive behavior: it was the object’s appearance and/or the fact that it captured parent/child attention at the moment the word was uttered, not the presence of the object throughout the video, that predicted observers’ accuracy. Study 2 experimentally investigated the precision of the timing relation, and whether it has an effect on observer accuracy, by disrupting the timing between when the word was uttered and the behaviors present in the videos as they were originally recorded. Disrupting timing by only +/− 1 to 2 sec. reduced participant confidence and significantly decreased their accuracy in word identification. The results enhance an expanding literature on how dyadic attentional factors can influence early vocabulary growth. By hypothesis, this kind of time-sensitive data-selection process operates as a filter on input, removing many extraneous and ill-supported word-meaning hypotheses from consideration during children’s early vocabulary learning.

Denitrifying NO reductases are evolutionarily related to the superfamily of heme-copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N2O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines, and Val-68 to a glutamic acid to create a nonheme FeB site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV-vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and CuI- or ZnII-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}7 species observable at liquid nitrogen temperature, the FeII-loaded proteins are EPR silent at ≥ 30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using 15NO and 15N18O. The apo- and CuI-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of FeII at the FeB site shifts the heme ν(FeNO) by +17 cm-1 and the ν(NO) by -50 cm-1 to 1549 cm-1. This low ν(NO) is without precedent for a six-coordinate heme {FeNO}7 species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme FeII. Detection of a similarly low ν(NO) in the ZnII-loaded protein supports this interpretation.

A conserved 2-His-1-Glu metal center, as found in natural non-heme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe B Mb(-His)). A high resolution (1.65 Å) crystal structure of Cu(II)-CN − -Fe B Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe B Mb(-His) can bind Cu, Fe or Zn ions, with both Cu(I)-Fe B Mb(-His) and Fe(II)-Fe B Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal yi-lu@illinois.edu . Δ Present address: