Rare antibody phage isolation and discrimination (RAPID) biopanning enables identification of high-affinity antibodies against challenging targets.

In vitro biopanning platforms using synthetic phage display antibody libraries have enabled the identification of antibodies against antigens that were once thought to be beyond the scope of immunization. Applying these methods against challenging targets remains a critical challenge. Here, we present a new biopanning pipeline, RAPID (Rare Antibody Phage Isolation and Discrimination), for the identification of rare high-affinity antibodies against challenging targets. RAPID biopanning uses fluorescent labeled phage displayed fragment antigen-binding (Fab) antibody libraries for the isolation of high-affinity binders with fluorescent activated sorting. Subsequently, discriminatory hit screening is performed with a biolayer interferometry (BLI) method, BIAS (Biolayer Interferometry Antibody Screen), where candidate binders are ranked and prioritized according to their estimated kinetic off rates. Previously reported antibodies were used to develop the methodology, and the RAPID biopanning pipeline was applied to three challenging targets (CHIP, Gαq, and CS3D), enabling the identification of high-affinity antibodies.

Promiscuity of response regulators for thioredoxin steers bacterial virulence.

The exquisite specificity between a sensor kinase and its cognate response regulator ensures faithful partner selectivity within two-component pairs concurrently firing in a single bacterium, minimizing crosstalk with other members of this conserved family of paralogous proteins. We show that conserved hydrophobic and charged residues on the surface of thioredoxin serve as a docking station for structurally diverse response regulators. Using the OmpR protein, we identify residues in the flexible linker and the C-terminal ß-hairpin that enable associations of this archetypical response regulator with thioredoxin, but are dispensable for interactions of this transcription factor to its cognate sensor kinase EnvZ, DNA or RNA polymerase. Here we show that the promiscuous interactions of response regulators with thioredoxin foster the flow of information through otherwise highly dedicated two-component signaling systems, thereby enabling both the transcription of Salmonella pathogenicity island-2 genes as well as growth of this intracellular bacterium in macrophages and mice.

Ligand-specific conformational change drives interdomain allostery in Pin1.

Pin1 is a two-domain cell regulator that isomerizes peptidyl-prolines. The catalytic domain (PPIase) and the other ligand-binding domain (WW) sample extended and compact conformations. Ligand binding changes the equilibrium of the interdomain conformations, but the conformational changes that lead to the altered domain sampling were unknown. Prior evidence has supported an interdomain allosteric mechanism. We recently introduced a magnetic resonance-based protocol that allowed us to determine the coupling of intra- and interdomain structural sampling in apo Pin1. Here, we describe ligand-specific conformational changes that occur upon binding of pCDC25c and FFpSPR. pCDC25c binding doubles the population of the extended states compared to the virtually identical populations of the apo and FFpSPR-bound forms. pCDC25c binding to the WW domain triggers conformational changes to propagate via the interdomain interface to the catalytic site, while FFpSPR binding displaces a helix in the PPIase that leads to repositioning of the PPIase catalytic loop.

On the use of residual dipolar couplings in multi-state structure calculation of two-domain proteins.

Residual dipolar couplings (RDCs) are powerful nuclear magnetic resonance (NMR) probes for the structure calculation of biomacromolecules. Typically, an alignment tensor that defines the orientation of the entire molecule relative to the magnetic field is determined either before refinement of individual bond vectors or simultaneously with this refinement. For single-domain proteins this approach works well since all bond vectors can be described within the same coordinate frame, which is given by the alignment tensor. However, novel approaches are sought after for systems where no universal alignment tensor can be used. Here, we present an approach that can be applied to two-domain proteins that enables the calculation of multiple states within each domain as well as with respect to the relative positions of the two domains.

Reconstruction of Coupled Intra- and Interdomain Protein Motion from Nuclear and Electron Magnetic Resonance.

Proteins composed of multiple domains allow for structural heterogeneity and interdomain dynamics that may be vital for function. Intradomain structures and dynamics can influence interdomain conformations and vice versa. However, no established structure determination method is currently available that can probe the coupling of these motions. The protein Pin1 contains separate regulatory and catalytic domains that sample "extended" and "compact" states, and ligand binding changes this equilibrium. Ligand binding and interdomain distance have been shown to impact the activity of Pin1, suggesting interdomain allostery. In order to characterize the conformational equilibrium of Pin1, we describe a novel method to model the coupling between intra- and interdomain dynamics at atomic resolution using multistate ensembles. The method uses time-averaged nuclear magnetic resonance (NMR) restraints and double electron-electron resonance (DEER) data that resolve distance distributions. While the intradomain calculation is primarily driven by exact nuclear Overhauser enhancements (eNOEs), J couplings, and residual dipolar couplings (RDCs), the relative domain distribution is driven by paramagnetic relaxation enhancement (PREs), RDCs, interdomain NOEs, and DEER. Our data support a 70:30 population of the compact and extended states in apo Pin1. A multistate ensemble describes these conformations simultaneously, with distinct conformational differences located in the interdomain interface stabilizing the compact or extended states. We also describe correlated conformations between the catalytic site and interdomain interface that may explain allostery driven by interdomain contact.

The Inherent Dynamics and Interaction Sites of the SARS-CoV-2 Nucleocapsid N-Terminal Region.

The nucleocapsid protein is one of four structural proteins encoded by SARS-CoV-2 and plays a central role in packaging viral RNA and manipulating the host cell machinery, yet its dynamic behavior and promiscuity in nucleotide binding has made standard structural methods to address its atomic-resolution details difficult. To begin addressing the SARS-CoV-2 nucleocapsid protein interactions with both RNA and the host cell along with its dynamic behavior, we have specifically focused on the folded N-terminal domain (NTD) and its flanking regions using nuclear magnetic resonance solution studies. Studies performed here reveal a large repertoire of interactions, which includes a temperature-dependent self-association mediated by the disordered flanking regions that also serve as binding sites for host cell cyclophilin-A while nucleotide binding is largely mediated by the central NTD core. NMR studies that include relaxation experiments have revealed the complicated dynamic nature of this viral protein. Specifically, while much of the N-terminal core domain exhibits micro-millisecond motions, a central ß-hairpin shows elevated inherent flexibility on the pico-nanosecond timescale and the serine/arginine-rich region of residues 176-209 undergoes multiple exchange phenomena. Collectively, these studies have begun to reveal the complexities of the nucleocapsid protein dynamics and its preferred interaction sites with its biological targets.

The Disordered Spindly C-terminus Interacts with RZZ Subunits ROD-1 and ZWL-1 in the Kinetochore through the Same Sites in C. Elegans.

Spindly is a dynein adaptor involved in chromosomal segregation during cell division. While Spindly's N-terminal domain binds to the microtubule motor dynein and its activator dynactin, the C-terminal domain (Spindly-C) binds its cargo, the ROD/ZW10/ZWILCH (RZZ) complex in the outermost layer of the kinetochore. In humans, Spindly-C binds to ROD, while in C. elegans Spindly-C binds to both Zwilch (ZWL-1) and ROD-1. Here, we employed various biophysical techniques to characterize the structure, dynamics and interaction sites of C. elegans Spindly-C. We found that despite the overall disorder, there are two regions with variable α-helical propensity. One of these regions is located in the C-terminal half and is compact; the second is sparsely populated in the N-terminal half. The interactions with both ROD-1 and ZWL-1 are mostly mediated by the same two sequentially remote disordered segments of Spindly-C, which are C-terminally adjacent to the helical regions. The findings suggest that the Spindly-C binding sites on ROD-1 in the ROD-1/ZWL-1 complex context are either shielded or conformationally weakened by the presence of ZWL-1 such that only ZWL-1 directly interacts with Spindly-C in C. elegans.

Reducing the measurement time of exact NOEs by non-uniform sampling.

We have previously reported on the measurement of exact NOEs (eNOEs), which yield a wealth of additional information in comparison to conventional NOEs. We have used these eNOEs in a variety of applications, including calculating high-resolution structures of proteins and RNA molecules. The collection of eNOEs is challenging, however, due to the need to measure a NOESY buildup series consisting of typically four NOESY spectra with varying mixing times in a single measurement session. While the 2D version can be completed in a few days, a fully sampled 3D-NOESY buildup series can take 10 days or more to acquire. This can be both expensive as well as problematic in the case of samples that are not stable over such a long period of time. One potential method to significantly decrease the required measurement time of eNOEs is to use non-uniform sampling (NUS) to decrease the number of points measured in the indirect dimensions. The effect of NUS on the extremely tight distance restraints extracted from eNOEs may be very pronounced. Therefore, we investigated the fidelity of eNOEs measured from three test cases at decreasing NUS densities: the 18.4 kDa protein human Pin1, the 4.1 kDa WW domain of Pin1 (both in 3D), and a 4.6 kDa 14mer RNA UUCG tetraloop (2D). Our results show that NUS imparted negligible error on the eNOE distances derived from good quality data down to 10% sampling for all three cases, but there is a noticeable decrease in the eNOE yield that is dependent upon the underlying sparsity, and thus complexity, of the sample. For Pin1, this transition occurred at roughly 40% while for the WW domain and the UUCG tetraloop it occurred at lower NUS densities of 20% and 10%, respectively. We rationalized these numbers through reconstruction simulations under various conditions. The extent of this loss depends upon the number of scans taken as well as the number of peaks to be reconstructed. Based on these findings, we have created guidelines for choosing an optimal NUS density depending on the number of peaks needed to be reconstructed in the densest region of a 2D or 3D NOESY spectrum.

Activity and Affinity of Pin1 Variants.

Pin1 is a peptidyl-prolyl isomerase responsible for isomerizing phosphorylated S/T-P motifs. Pin1 has two domains that each have a distinct ligand binding site, but only its PPIase domain has catalytic activity. Vast evidence supports interdomain allostery of Pin1, with binding of a ligand to its regulatory WW domain impacting activity in the PPIase domain. Many diverse studies have made mutations in Pin1 in order to elucidate interactions that are responsible for ligand binding, isomerase activity, and interdomain allostery. Here, we summarize these mutations and their impact on Pin1's structure and function.

Solution NMR backbone assignment reveals interaction-free tumbling of human lineage-specific Olduvai protein domains.

Olduvai protein domains, encoded primarily by NBPF genes, have been linked to both human brain evolution and cognitive diseases such as autism and schizophrenia. There are six primary domains that comprise the Olduvai family: three conserved domains (CON1-3) and three human lineage-specific domains (HLS1-3), which typically occur as a triplet (HLS1, HLS2 and HLS3). Herein, we present the solution NMR assignment of the backbone chemical shifts of the separate HLS1, 2 and 3 domains of NBPF15. Our data suggest that there is no change in the structure of the separate domains when compared to the full-length triplet (HLS1-HLS2-HLS3). We also demonstrate that there is no direct interaction between the three domains.

A transient helix in the disordered region of dynein light intermediate chain links the motor to structurally diverse adaptors for cargo transport.

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.

Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery.

Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain 1H, 13C and 15N NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.

High-resolution small RNA structures from exact nuclear Overhauser enhancement measurements without additional restraints.

RNA not only translates the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by nuclear magnetic resonance (NMR). However, as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely obtained from nuclear Overhauser enhancement (NOE) data alone. Instead, additional semi-empirical restraints and labor-intensive techniques are required for structural averages, while there are only a few experimentally derived ensembles representing dynamics. Here we show that our exact NOE (eNOE) based structure determination protocol is able to define a 14-mer UUCG tetraloop structure at high resolution without other restraints. Additionally, we use eNOEs to calculate a two-state structure, which samples its conformational space. The protocol may open an avenue to obtain high-resolution structures of small RNA of unprecedented accuracy with moderate experimental efforts.

Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA.

Distance-dependent nuclear Overhauser enhancements (NOEs) are one of the most popular and important experimental restraints for calculating NMR structures. Despite this, they are mostly employed as semiquantitative upper distance bounds, and this discards the wealth of information that is encoded in the cross-relaxation rate constant. Information that is lost includes exact distances between protons and dynamics that occur on the sub-millisecond timescale. Our recently introduced exact measurement of the NOE (eNOE) requires little additional experimental effort relative to other NMR observables. So far, we have used eNOEs to calculate multistate ensembles of proteins up to approximately 150 residues. Here, we briefly revisit eNOE methodology and present two new directions for the use of eNOEs: applications to large proteins and RNA.

Efficient Stereospecific Hß2/3 NMR Assignment Strategy for Mid-Size Proteins.

We present a strategy for stereospecific NMR assignment of Hß2 and Hß3 protons in mid-size proteins (~150 residues). For such proteins, resonance overlap in standard experiments is severe, thereby preventing unambiguous assignment of a large fraction of ß-methylenes. To alleviate this limitation, assignment experiments may be run in high static fields, where higher decoupling power is required. Three-bond Hα-Hß J-couplings (3 J Hα-Hß) are critical for stereospecific assignments of ß-methylene protons, and for determining rotameric χ1 states. Therefore, we modified a pulse sequence designed to measure accurate 3 J Hα-Hß couplings such that probe heating was reduced, while the decoupling performance was improved. To further increase the resolution, we applied non-uniform sampling (NUS) schemes in the indirect 1H and 13C dimensions. The approach was applied to two medium-sized proteins, odorant binding protein 22 (OBP22; 14.4 kDa) and Pin1 (18.2 kDa), at 900 MHz polarizing fields. The coupling values obtained from NUS and linear sampling were extremely well correlated. However, NUS decreased the overlap of Hß2/3 protons, thus supplying a higher yield of extracted 3 J Hα-Hß coupling values when compared with linear sampling. A similar effect could be achieved with linear prediction applied to the linearly sampled data prior to the Fourier transformation. Finally, we used 3 J Hα-Hß couplings from Pin1 in combination with either conventional or exact nuclear Overhauser enhancement (eNOE) restraints to determine the stereospecific assignments of ß-methylene protons. The use of eNOEs further increased the fraction of unambiguously assigned resonances when compared with procedures using conventional NOEs.

The Exact Nuclear Overhauser Enhancement: Recent Advances.

Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.