Serodominant SARS-CoV-2 Nucleocapsid Peptides Map to Unstructured Protein Regions.

The SARS-CoV-2 nucleocapsid (N) protein is highly immunogenic, and anti-N antibodies are commonly used as markers for prior infection. While several studies have examined or predicted the antigenic regions of N, these have lacked consensus and structural context. Using COVID-19 patient sera to probe an overlapping peptide array, we identified six public and four private epitope regions across N, some of which are unique to this study. We further report the first deposited X-ray structure of the stable dimerization domain at 2.05 Å as similar to all other reported structures. Structural mapping revealed that most epitopes are derived from surface-exposed loops on the stable domains or from the unstructured linker regions. An antibody response to an epitope in the stable RNA binding domain was found more frequently in sera from patients requiring intensive care. Since emerging amino acid variations in N map to immunogenic peptides, N protein variation could impact detection of seroconversion for variants of concern. IMPORTANCE As SARS-CoV-2 continues to evolve, a structural and genetic understanding of key viral epitopes will be essential to the development of next-generation diagnostics and vaccines. This study uses structural biology and epitope mapping to define the antigenic regions of the viral nucleocapsid protein in sera from a cohort of COVID-19 patients with diverse clinical outcomes. These results are interpreted in the context of prior structural and epitope mapping studies as well as in the context of emergent viral variants. This report serves as a resource for synthesizing the current state of the field toward improving strategies for future diagnostic and therapeutic design.

Critical Assessment of Self-Consistency Checks in the All-Atom Molecular Dynamics Simulation of Intrinsically Disordered Proteins.

All atom simulations can be used to quantify conformational properties of Intrinsically Disordered Proteins (IDP). However, simulations must satisfy convergence checks to ensure observables computed from simulation are reliable and reproducible. While absolute convergence is purely a theoretical concept requiring infinitely long simulation, a more practical, yet rigorous, approach is to impose Self Consistency Checks (SCCs) to gain confidence in the simulated data. Currently there is no study of SCCs in IDPs, unlike their folded counterparts. In this paper, we introduce different criteria for self-consistency checks for IDPs. Next, we impose these SCCs to critically assess the performance of different simulation protocols using the N terminal domain of HIV Integrase and the linker region of SARS-CoV-2 Nucleoprotein as two model IDPs. All simulation protocols begin with all-atom implicit solvent Monte Carlo (MC) simulation and subsequent clustering of MC generated conformations to create the representative structures of the IDPs. These representative structures serve as the initial structure for subsequent molecular dynamics (MD) runs with explicit solvent. We conclude that generating multiple short (∼3 µs) MD simulation trajectories─all starting from the most representative MC generated conformation─and merging them is the protocol of choice due to (i) its ability to satisfy multiple SCCs, (ii) consistently reproducing experimental data, and (iii) the efficiency of running independent trajectories in parallel by harnessing multiple cores available in modern GPU clusters. Running one long trajectory (greater than 20 µs) can also satisfy the first two criteria but is less desirable due to prohibitive computation time. These findings help resolve the challenge of identifying a usable starting configuration, provide an objective measure of SCC, and establish rigorous criteria to determine the minimum length (for one long simulation) or number of trajectories needed in all-atom simulation of IDPs.

Liaisons dangereuses: Intrinsic Disorder in Cellular Proteins Recruited to Viral Infection-Related Biocondensates.

Liquid-liquid phase separation (LLPS) is responsible for the formation of so-called membrane-less organelles (MLOs) that are essential for the spatio-temporal organization of the cell. Intrinsically disordered proteins (IDPs) or regions (IDRs), either alone or in conjunction with nucleic acids, are involved in the formation of these intracellular condensates. Notably, viruses exploit LLPS at their own benefit to form viral replication compartments. Beyond giving rise to biomolecular condensates, viral proteins are also known to partition into cellular MLOs, thus raising the question as to whether these cellular phase-separating proteins are drivers of LLPS or behave as clients/regulators. Here, we focus on a set of eukaryotic proteins that are either sequestered in viral factories or colocalize with viral proteins within cellular MLOs, with the primary goal of gathering organized, predicted, and experimental information on these proteins, which constitute promising targets for innovative antiviral strategies. Using various computational approaches, we thoroughly investigated their disorder content and inherent propensity to undergo LLPS, along with their biological functions and interactivity networks. Results show that these proteins are on average, though to varying degrees, enriched in disorder, with their propensity for phase separation being correlated, as expected, with their disorder content. A trend, which awaits further validation, tends to emerge whereby the most disordered proteins serve as drivers, while more ordered cellular proteins tend instead to be clients of viral factories. In light of their high disorder content and their annotated LLPS behavior, most proteins in our data set are drivers or co-drivers of molecular condensation, foreshadowing a key role of these cellular proteins in the scaffolding of viral infection-related MLOs.

Distinguishing features of fold-switching proteins.

Though many folded proteins assume one stable structure that performs one function, a small-but-increasing number remodel their secondary and tertiary structures and change their functions in response to cellular stimuli. These fold-switching proteins regulate biological processes and are associated with autoimmune dysfunction, severe acute respiratory syndrome coronavirus-2 infection, and more. Despite their biological importance, it is difficult to computationally predict fold switching. With the aim of advancing computational prediction and experimental characterization of fold switchers, this review discusses several features that distinguish fold-switching proteins from their single-fold and intrinsically disordered counterparts. First, the isolated structures of fold switchers are less stable and more heterogeneous than single folders but more stable and less heterogeneous than intrinsically disordered proteins (IDPs). Second, the sequences of single fold, fold switching, and intrinsically disordered proteins can evolve at distinct rates. Third, proteins from these three classes are best predicted using different computational techniques. Finally, late-breaking results suggest that single folders, fold switchers, and IDPs have distinct patterns of residue-residue coevolution. The review closes by discussing high-throughput and medium-throughput experimental approaches that might be used to identify new fold-switching proteins.

Intrinsically disordered proteins in viral pathogenesis and infections.

Disordered proteins serve a crucial part in many biological processes that go beyond the capabilities of ordered proteins. A large number of virus-encoded proteins have extremely condensed proteomes and genomes, which results in highly disordered proteins. The presence of these IDPs allows them to rapidly adapt to changes in their biological environment and play a significant role in viral replication and down-regulation of host defense mechanisms. Since viruses undergo rapid evolution and have a high rate of mutation and accumulation in their proteome, IDPs' insights into viruses are critical for understanding how viruses hijack cells and cause disease. There are many conformational changes that IDPs can adopt in order to interact with different protein partners and thus stabilize the particular fold and withstand high mutation rates. This chapter explains the molecular mechanism behind viral IDPs, as well as the significance of recent research in the field of IDPs, with the goal of gaining a deeper comprehension of the essential roles and functions played by viral proteins.

Exploring Manually Curated Annotations of Intrinsically Disordered Proteins with DisProt.

DisProt is the major repository of manually curated data for intrinsically disordered proteins collected from the literature. Although lacking a stable three-dimensional structure under physiological conditions, intrinsically disordered proteins carry out a plethora of biological functions, some of them directly arising from their flexible nature. A growing number of scientific studies have been published during the last few decades to shed light on their unstructured state, their binding modes, and their functions. DisProt makes use of a team of expert biocurators to provide up-to-date annotations of intrinsically disordered proteins from the literature, making them available to the scientific community. Here we present a comprehensive description on how to use DisProt in different contexts and provide a detailed explanation of how to explore and interpret manually curated annotations of intrinsically disordered proteins. We describe how to search DisProt annotations, both using the web interface and the API for programmatic access. Finally, we explain how to visualize and interpret a DisProt entry, the SARS-CoV-2 Nucleoprotein, characterized by the presence of unstructured N-terminal and C-terminal regions and a flexible linker. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Performing a search in DisProt Support Protocol 1: Downloading options Support Protocol 2: Programmatic access with DisProt REST API Basic Protocol 2: Exploring the DisProt Ontology page Basic Protocol 3: Visualizing and interpreting DisProt entries-the SARS-CoV-2 Nucleoprotein use case.

Modeling the Dynamics of Protein-Protein Interfaces, How and Why?

Protein-protein assemblies act as a key component in numerous cellular processes. Their accurate modeling at the atomic level remains a challenge for structural biology. To address this challenge, several docking and a handful of deep learning methodologies focus on modeling protein-protein interfaces. Although the outcome of these methods has been assessed using static reference structures, more and more data point to the fact that the interaction stability and specificity is encoded in the dynamics of these interfaces. Therefore, this dynamics information must be taken into account when modeling and assessing protein interactions at the atomistic scale. Expanding on this, our review initially focuses on the recent computational strategies aiming at investigating protein-protein interfaces in a dynamic fashion using enhanced sampling, multi-scale modeling, and experimental data integration. Then, we discuss how interface dynamics report on the function of protein assemblies in globular complexes, in fuzzy complexes containing intrinsically disordered proteins, as well as in active complexes, where chemical reactions take place across the protein-protein interface.

14-3-3 Proteins are Potential Regulators of Liquid-Liquid Phase Separation.

The 14-3-3 family proteins are vital scaffold proteins that ubiquitously expressed in various tissues. They interact with numerous protein targets and mediate many cellular signaling pathways. The 14-3-3 binding motifs are often embedded in intrinsically disordered regions which are closely associated with liquid-liquid phase separation (LLPS). In the past ten years, LLPS has been observed for a variety of proteins and biological processes, indicating that LLPS plays a fundamental role in the formation of membraneless organelles and cellular condensates. While extensive investigations have been performed on 14-3-3 proteins, its involvement in LLPS is overlooked. To date, 14-3-3 proteins have not been reported to undergo LLPS alone or regulate LLPS of their binding partners. To reveal the potential involvement of 14-3-3 proteins in LLPS, in this review, we summarized the LLPS propensity of 14-3-3 binding partners and found that about one half of them may undergo LLPS spontaneously. We further analyzed the phase separation behavior of representative 14-3-3 binders and discussed how 14-3-3 proteins may be involved. By modulating the conformation and valence of interactions and recruiting other molecules, we speculate that 14-3-3 proteins can efficiently regulate the functions of their targets in the context of LLPS. Considering the critical roles of 14-3-3 proteins, there is an urgent need for investigating the involvement of 14-3-3 proteins in the phase separation process of their targets and the underling mechanisms.

Intrinsically Disordered Proteins: Perspective on COVID-19 Infection and Drug Discovery.

Since the beginning of the COVID-19 pandemic caused by SARS-CoV-2, millions of patients have been diagnosed and many of them have died from the disease worldwide. The identification of novel therapeutic targets are of utmost significance for prevention and treatment of COVID-19. SARS-CoV-2 is a single-stranded RNA virus with a 30 kb genome packaged into a membrane-enveloped virion, transcribing several tens of proteins. The belief that the amino acid sequence of proteins determines their 3D structure which, in turn, determines their function has been a central principle of molecular biology for a long time. Recently, it has been increasingly realized, however, that there is a large group of proteins that lack a fixed or ordered 3D structure, yet they exhibit important biological activities─so-called intrinsically disordered proteins and protein regions (IDPs/IDRs). Disordered regions in viral proteins are generally associated with viral infectivity and pathogenicity because they endow the viral proteins the ability to easily and promiscuously bind to host proteins; therefore, the proteome of SARS-CoV-2 has been thoroughly examined for intrinsic disorder. It has been recognized that, in fact, the SARS-CoV-2 proteome exhibits significant levels of structural order, with only the nucleocapsid (N) structural protein and two of the nonstructural proteins being highly disordered. The spike (S) protein of SARS-CoV-2 exhibits significant levels of structural order, yet its predicted percentage of intrinsic disorder is still higher than that of the spike protein of SARS-CoV. Noteworthy, however, even though IDPs/IDRs are not common in the SARS-CoV-2 proteome, the existing ones play major roles in the functioning and virulence of the virus and are thus promising drug targets for rational antiviral drug design. Presented here is a COVID-19 perspective on the intrinsically disordered proteins, summarizing recent results on the SARS-CoV-2 proteome disorder features, their physiological and pathological relevance, and their prominence as prospective drug target sites.

SARS-CoV-2 variants preferentially emerge at intrinsically disordered protein sites helping immune evasion.

The SARS-CoV-2 pandemic is maintained by the emergence of successive variants, highlighting the flexibility of the protein sequences of the virus. We show that experimentally determined intrinsically disordered regions (IDRs) are abundant in the SARS-CoV-2 viral proteins, making up to 28% of disorder content for the S1 subunit of spike and up to 51% for the nucleoprotein, with the vast majority of mutations occurring in the 13 major variants mapped to these IDRs. Strikingly, antigenic sites are enriched in IDRs, in the receptor-binding domain (RBD) and in the N-terminal domain (NTD), suggesting a key role of structural flexibility in the antigenicity of the SARS-CoV-2 protein surface. Mutations occurring in the S1 subunit and nucleoprotein (N) IDRs are critical for immune evasion and antibody escape, suggesting potential additional implications for vaccines and monoclonal therapeutic strategies. Overall, this suggests the presence of variable regions on S1 and N protein surfaces, which confer sequence and antigenic flexibility to the virus without altering its protein functions.

Dynamic, but Not Necessarily Disordered, Human-Virus Interactions Mediated through SLiMs in Viral Proteins.

Most viruses have small genomes that encode proteins needed to perform essential enzymatic functions. Across virus families, primary enzyme functions are under functional constraint; however, secondary functions mediated by exposed protein surfaces that promote interactions with the host proteins may be less constrained. Viruses often form transient interactions with host proteins through conformationally flexible interfaces. Exposed flexible amino acid residues are known to evolve rapidly suggesting that secondary functions may generate diverse interaction potentials between viruses within the same viral family. One mechanism of interaction is viral mimicry through short linear motifs (SLiMs) that act as functional signatures in host proteins. Viral SLiMs display specific patterns of adjacent amino acids that resemble their host SLiMs and may occur by chance numerous times in viral proteins due to mutational and selective processes. Through mimicry of SLiMs in the host cell proteome, viruses can interfere with the protein interaction network of the host and utilize the host-cell machinery to their benefit. The overlap between rapidly evolving protein regions and the location of functionally critical SLiMs suggest that these motifs and their functional potential may be rapidly rewired causing variation in pathogenicity, infectivity, and virulence of related viruses. The following review provides an overview of known viral SLiMs with select examples of their role in the life cycle of a virus, and a discussion of the structural properties of experimentally validated SLiMs highlighting that a large portion of known viral SLiMs are devoid of predicted intrinsic disorder based on the viral SLiMs from the ELM database.

MemDis: Predicting Disordered Regions in Transmembrane Proteins.

Transmembrane proteins (TMPs) play important roles in cells, ranging from transport processes and cell adhesion to communication. Many of these functions are mediated by intrinsically disordered regions (IDRs), flexible protein segments without a well-defined structure. Although a variety of prediction methods are available for predicting IDRs, their accuracy is very limited on TMPs due to their special physico-chemical properties. We prepared a dataset containing membrane proteins exclusively, using X-ray crystallography data. MemDis is a novel prediction method, utilizing convolutional neural network and long short-term memory networks for predicting disordered regions in TMPs. In addition to attributes commonly used in IDR predictors, we defined several TMP specific features to enhance the accuracy of our method further. MemDis achieved the highest prediction accuracy on TMP-specific dataset among other popular IDR prediction methods.

Partial structure, dampened mobility, and modest impact of a His tag in the SARS-CoV-2 Nsp2 C-terminal region.

Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many viruses use their own IDPs to "hack" these processes to deactivate host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational preferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2, whose C-terminal region (CtR) is predicted to be disordered, interacts with human proteins that regulate translation initiation and endosome vesicle sorting. Molecules that block these interactions could be valuable leads for drug development. The 13Cß and backbone 13CO, 1HN, 13Cα, and 15N nuclei of Nsp2's 45-residue CtR were assigned and used to characterize its structure and dynamics in three contexts; namely: (1) retaining an N-terminal His tag, (2) without the His tag and with an adventitious internal cleavage, and (3) lacking both the His tag and the internal cleavage. Two five-residue segments adopting a minor extended population were identified. Overall, the dynamic behavior is midway between a completely rigid and a fully flexible chain. Whereas the presence of an N-terminal His tag and internal cleavage stiffen and loosen, respectively, neighboring residues, they do not affect the tendency of two regions to populate extended conformations.

Protein Intrinsic Disorder and Evolvability of MERS-CoV.

Middle East Respiratory Syndrome (MERS) is a viral respiratory disease caused by one of the human coronaviruses, MERS-CoV [...].

The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging. Therefore, typically employed purification methods appear to be insufficient for removing nucleic acid contamination. In this study, we present a novel purification protocol that enables the N protein to be prepared without any bound nucleic acids. We also performed comparative structural analysis of the N protein contaminated with nucleic acids and free of contamination and showed significant differences in the structural and phase separation properties of the protein. These results indicate that nucleic-acid contamination may severely affect molecular properties of the purified N protein. In addition, the notable ability of the N protein to form condensates whose morphology and behaviour suggest more ordered forms resembling gel-like or solid structures is described.

Tackling Covid-19 using disordered-to-order transition of residues in the spike protein upon angiotensin-converting enzyme 2 binding.

The 2019-novel coronavirus also known as severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a common threat to animals and humans, and is responsible for the human SARS pandemic in 2019 to 2021. The infection of SARS-CoV-2 in humans involves a viral surface glycoprotein named as spike proteins, which bind to the human angiotensin-converting enzyme 2 (ACE2) proteins. Particularly, the receptor binding domains (RBDs) mediate the interaction and contain several disordered regions, which help in the binding. Investigations on the influence of disordered residues/regions in stability and binding of spike protein with ACE2 help to understand the disease pathogenesis, which has not yet been studied. In this study, we have used molecular-dynamics simulations to characterize the structural changes in disordered regions of the spike protein that result from ACE2 binding. We observed that the disordered regions undergo disorder-to-order transition (DOT) upon binding with ACE2, and the DOT residues are located at functionally important regions of RBD. Although the RBD is having rigid structure, DOT residues make conformational rearrangements for the spike protein to attach with ACE2. The binding is strengthened via hydrophilic and aromatic amino acids mainly present in the DOTs. The positively correlated motions of the DOT residues with its nearby residues also explain the binding profile of RBD with ACE2, and the residues are observed to be contributing more favorable binding energies for the spike-ACE2 complex formation. This study emphasizes that intrinsically disordered residues in the RBD of spike protein may provide insights into its etiology and be useful for drug and vaccine discovery.

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications.

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.

A Novel Strategy for the Development of Vaccines for SARS-CoV-2 (COVID-19) and Other Viruses Using AI and Viral Shell Disorder.

A model that predicts levels of coronavirus (CoV) respiratory and fecal-oral transmission potentials based on the shell disorder has been built using neural network (artificial intelligence, AI) analysis of the percentage of disorder (PID) in the nucleocapsid, N, and membrane, M, proteins of the inner and outer viral shells, respectively. Using primarily the PID of N, SARS-CoV-2 is grouped as having intermediate levels of both respiratory and fecal-oral transmission potentials. Related studies, using similar methodologies, have found strong positive correlations between virulence and inner shell disorder among numerous viruses, including Nipah, Ebola, and Dengue viruses. There is some evidence that this is also true for SARS-CoV-2 and SARS-CoV, which have N PIDs of 48% and 50%, and case-fatality rates of 0.5-5% and 10.9%, respectively. The underlying relationship between virulence and respiratory potentials has to do with the viral loads of vital organs and body fluids, respectively. Viruses can spread by respiratory means only if the viral loads in saliva and mucus exceed certain minima. Similarly, a patient is likelier to die when the viral load overwhelms vital organs. Greater disorder in inner shell proteins has been known to play important roles in the rapid replication of viruses by enhancing the efficiency pertaining to protein-protein/DNA/RNA/lipid bindings. This paper suggests a novel strategy in attenuating viruses involving comparison of disorder patterns of inner shells (N) of related viruses to identify residues and regions that could be ideal for mutation. The M protein of SARS-CoV-2 has one of the lowest M PID values (6%) in its family, and therefore, this virus has one of the hardest outer shells, which makes it resistant to antimicrobial enzymes in body fluid. While this is likely responsible for its greater contagiousness, the risks of creating an attenuated virus with a more disordered M are discussed.

Shell Disorder Analysis Suggests That Pangolins Offered a Window for a Silent Spread of an Attenuated SARS-CoV-2 Precursor among Humans.

A model to predict the relative levels of respiratory and fecal-oral transmission potentials of coronaviruses (CoVs) by measuring the percentage of protein intrinsic disorder (PID) of the M (Membrane) and N (Nucleoprotein) proteins in their outer and inner shells, respectively, was built before the MERS-CoV outbreak. With MPID = 8.6% and NPID = 50.2%, the 2003 SARS-CoV falls into group B, which consists of CoVs with intermediate levels of both fecal-oral and respiratory transmission potentials. Further validation of the model came with MERS-CoV (MPID = 9%, NPID = 44%) and SARS-CoV-2 (MPID = 5.5%, NPID = 48%) falling into the groups C and B, respectively. Group C contains CoVs with higher fecal-oral but lower respiratory transmission potentials. Unlike SARS-CoV, SARS-CoV-2 with MPID = 5.5% has one of the hardest outer shells among CoVs. Because the hard shell is able to resist the antimicrobial enzymes in body fluids, the infected person is able to shed large quantities of viral particles via saliva and mucus, which could account for the higher contagiousness of SARS-COV-2. Further searches have found that high rigidity of the outer shell is characteristic for the CoVs of burrowing animals, such as rabbits (MPID = 5.6%) and pangolins (MPID = 5-6%), which are in contact with the buried feces. A closer inspection of pangolin-CoVs from 2017 to 2019 reveals that pangolins provided a unique window of opportunity for the entry of an attenuated SARS-CoV-2 precursor into the human population in 2017 or earlier, with the subsequent slow and silent spread as a mild cold that followed by its mutations into the current more virulent form. Evidence of this lies in both the genetic proximity of the pangolin-CoVs to SARS-CoV-2 (∼90%) and differences in N disorder. A 2017 pangolin-CoV strain shows evidence of higher levels of attenuation and higher fecal-oral transmission associated with lower human infectivity via having lower NPID (44.8%). Our shell disorder model predicts this to be a SARS-CoV-2 vaccine strain, as lower inner shell disorder is associated with the lesser virulence in a variety of viruses.

Understanding COVID-19 via comparative analysis of dark proteomes of SARS-CoV-2, human SARS and bat SARS-like coronaviruses.

The recently emerged coronavirus designated as SARS-CoV-2 (also known as 2019 novel coronavirus (2019-nCoV) or Wuhan coronavirus) is a causative agent of coronavirus disease 2019 (COVID-19), which is rapidly spreading throughout the world now. More than 1.21 million cases of SARS-CoV-2 infection and more than 67,000 COVID-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. The World Health Organization (WHO) has declared the SARS-CoV-2 spread as a global public health emergency and admitted COVID-19 as a pandemic now. Multiple sequence alignment data correlated with the already published reports on SARS-CoV-2 evolution indicated that this virus is closely related to the bat severe acute respiratory syndrome-like coronavirus (bat SARS-like CoV) and the well-studied human SARS coronavirus (SARS-CoV). The disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. Therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of SARS-CoV-2, bat SARS-like, and human SARS CoVs by analysing the prevalence of intrinsic disorder in their proteins. According to our findings, SARS-CoV-2 proteome contains very significant levels of structural order. In fact, except for nucleocapsid, Nsp8, and ORF6, the vast majority of SARS-CoV-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (IDPRs). However, IDPRs found in SARS-CoV-2 proteins are functionally important. For example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all SARS-CoV-2 proteins contains molecular recognition features (MoRFs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. The results of our extensive investigation of the dark side of SARS-CoV-2 proteome will have important implications in understanding the structural and non-structural biology of SARS or SARS-like coronaviruses.