Structure-function relationships in proteins are predicated on the spatial proximity of noncovalently interacting groups of atoms. Thus, structural elements located away from a protein's active site are typically presumed to serve a stabilizing or scaffolding role for the larger structure. Here we report a functional role for a distal structural element in a PDZ domain, even though it is not required to maintain PDZ structure. The third PDZ domain from PSD-95/SAP90 (PDZ3) has an unusual additional third alpha helix (␣3) that packs in contiguous fashion against the globular domain. Although ␣3 lies outside the active site and does not make direct contact with C-terminal peptide ligand, removal of ␣3 reduces ligand affinity by 21-fold. Further investigation revealed that the difference in binding free energies between the full-length and truncated constructs is predominantly entropic in nature and that without ␣3, picosecond-nanosecond side-chain dynamics are enhanced throughout the domain, as determined by 2 H methyl NMR relaxation. Thus, the distal modulation of binding function appears to occur via a delocalized conformational entropy mechanism. Without removal of ␣3 and characterization of side-chain dynamics, this dynamic allostery would have gone unnoticed. Moreover, what appeared at first to be an artificial modification of PDZ3 has been corroborated by experimentally verified phosphorylation of ␣3, revealing a tangible biological mechanism for this novel regulatory scheme. This hidden dynamic allostery raises the possibility of as-yet unidentified or untapped allosteric regulation in this PDZ domain and is a very clear example of function arising from dynamics rather than from structure.NMR ͉ PSD-95 ͉ spin relaxation ͉ entropy P roteins owe their functionality to the 3-dimensional arrangement of atoms. A typical protein's structure stabilizes its active site, allowing for specific interactions with substrate or ligand. These basic structure-function relationships are well understood for countless types of proteins. Because most active sites are relatively small, it has been presumed that the remaining bulk of the globular structure provides a scaffolding role. Thus, even though similar domains belonging to the same family may have substrate specificity preferences, the folds of those domains are composed of invariant structural elements (1). Nonetheless, variations in tertiary fold composition, such as additional elements of secondary structure, are not uncommon. An example of this can be seen within the PDZ domain family of proteins. From this, the question of whether there is a specific role for such auxiliary structural elements remains open. In other words, how might these additional elements influence the core domain?PDZ domains (eg, PSD-95, Discs Large, Zo-1) are small, Ϸ90-aa modular structures that typically bind C-terminal tails (Ϸ4-6 residues) of target proteins (2). They are frequently found in multiple copies in proteins with diverse functions, especially those involved in signal transduction c...

The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of (3)H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion.

The periodic emergence of novel coronaviruses (CoVs) represents an ongoing public health concern with significant health and financial burden worldwide. The most recent occurrence originated in the city of Wuhan, China where a novel coronavirus (SARS-CoV-2) emerged causing severe respiratory illness and pneumonia. The continual emergence of novel coronaviruses underscores the importance of developing effective vaccines as well as novel therapeutic options that target either viral functions or host factors recruited to support coronavirus replication. The CoV nonstructural protein 1 (nsp1) has been shown to promote cellular mRNA degradation, block host cell translation, and inhibit the innate immune response to virus infection. Interestingly, deletion of the nsp1-coding region in infectious clones prevented the virus from productively infecting cultured cells. Because of nsp1’s importance in the CoV life cycle, it has been highlighted as a viable target for both antiviral therapy and vaccine development. However, the fundamental molecular and structural mechanisms that underlie nsp1 function remain poorly understood, despite its critical role in the viral life cycle. Here we report the high-resolution crystal structure of the amino, globular portion of SARS-CoV-2 nsp1 (residues 10 – 127) at 1.77 Å resolution. A comparison of our structure with the SARS-CoV-1 nsp1 structure reveals how mutations alter the conformation of flexible loops, inducing the formation of novel secondary structural elements and new surface features. Paired with the recently published structure of the carboxyl end of nsp1 (residues 148 – 180), our results provide the groundwork for future studies focusing on SARS-CoV-2 nsp1 structure and function during the viral life cycle. IMPORTANCE The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. One protein known to play a critical role in the coronavirus life cycle is nonstructural protein1 (nsp1). As such, it has been highlighted in numerous studies as a target for both the development of antivirals and for the design of live-attenuated vaccines. Here we report the high-resolution crystal structure of nsp1 derived from SARS-CoV-2 at 1.77 Å resolution. This structure will facilitate future studies focusing on understanding the relationship between structure and function for nsp1. In turn, understanding these structure-function relationships will allow nsp1 to be fully exploited as a target for both antiviral development and vaccine design.

Postsynaptic density-95 is a multidomain scaffolding protein that recruits glutamate receptors to postsynaptic sites and facilitates signal processing and connection to the cytoskeleton. It is the leading member of the membrane-associated guanylate kinase family of proteins, which are defined by the PSD-95/ Discs large/ZO-1 (PDZ)-Src homology 3 (SH3)-guanylate kinase domain sequence. We used NMR to show that phosphorylation of conserved tyrosine 397, which occurs in vivo and is located in an atypical helical extension (␣3), initiates a rapid equilibrium of docked and undocked conformations. Undocking reduced ligand binding affinity allosterically and weakened the interaction of PDZ3 with SH3 even though these domains are separated by a ϳ25-residue linker. Additional phosphorylation at two linker sites further disrupted the interaction, implicating ␣3 and the linker in tuning interdomain communication. These experiments revealed a novel mode of regulation by a detachable PDZ element and offer a first glimpse at the dynamic interaction of PDZ and SH3-guanylate kinase domains in membrane-associated guanylate kinases.Phosphorylation is one of the most common modifications made to proteins. Depending on the protein substrate, it can regulate the gain or loss of activity through a variety of mechanisms. Most mechanisms studied to date appear to work via a change in steric geometry at the active site or a global conformational change in the protein (1-3), although a few recent examples indicate modulation of protein dynamics that can yield a graded response (4, 5). In some cases, phosphorylation simply provides a protein-protein recognition site (6). Overall, the extent of structural characterization of phosphorylation mechanisms is limited given the ubiquity of this post-translational signaling mechanism and the large diversity of effects observed upon phosphorylation. A greater understanding of how phosphorylation(s) modulates protein activity is essential for a mechanistic understanding of signal transduction.Many phosphorylated proteins are multidomain, modular proteins (2-4). One class of such proteins that shape signal transduction is the scaffolding proteins, which are frequently phosphorylated (7). Membrane-associated guanylate kinase (MAGUK) 2 family proteins are multidomain scaffolding proteins that play roles in ion channel clustering, intracellular trafficking, signal transduction, and cell-cell adhesion (8 -10). Malfunction of MAGUKs is associated with central nervous system disorders (11). The defining MAGUK signature is the PDZ-SH3-guanylate kinase "supradomain" architecture. The best known MAGUK protein is postsynaptic density-95 (PSD-95), which is found on the cytoplasmic side of postsynaptic terminals. PSD-95 is a non-catalytic scaffolding protein that clusters glutamate receptors and assembles macromolecular complexes for signal integration in the postsynapse at excitatory neurons (8,12). It consists of three PDZ domains, an SH3 domain, and a non-catalytic guanylate kinase (GK) domain (see Fig. 1A). ...

SignificanceIt is well known that ribosomal RNA processing is directly impacted by the rate of transcription elongation by RNA polymerase I (Pol I). To understand how these processes are orchestrated, we must carefully define transcription elongation properties in vitro and in living cells. Here, we characterize DNA sequence elements that pause and terminate Pol I transcription in vitro. We also establish methods for analyzing Pol I transcription elongation properties in vivo using native elongating transcript sequencing (NETSeq). Our NETSeq data revealed frequent pausing by Pol I and decreased Pol I occupancy at G residues, suggesting unequal rates of nucleotide incorporation by the enzyme. These findings redefine our understanding of Pol I transcription elongation and its heterogeneity in vivo.

Summary The influenza nonstructural protein 1 (NS1) plays a critical role in antagonizing the innate immune response to infection. One interaction that facilitates this function is between NS1 and RIG-I, one of the main sensors of influenza virus infection. While NS1 and RIG-I are known to interact, it is currently unclear whether this interaction is direct or if it is mediated by other biomolecules. In the present study, we demonstrate a direct, strain dependent interaction between the NS1 RNA binding domain (NS1RBD) of the influenza A/Brevig Mission/1918 H1N1 (1918H1N1) virus and the second CARD domain of RIG-I. Solving the solution structure of the 1918H1N1 NS1RBD revealed features in a functionally novel region that may facilitate the observed interaction. The biophysical and structural data herein suggest a possible mechanism by which strain specific differences in NS1 modulate influenza virulence.

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on 15N and 2H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in µs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect.