Design of High Affinity Binders to Convex Protein Target Sites.

While there has been progress in the de novo design of small globular miniproteins (50-65 residues) to bind to primarily concave regions of a target protein surface, computational design of minibinders to convex binding sites remains an outstanding challenge due to low level of overall shape complementarity. Here, we describe a general approach to generate computationally designed proteins which bind to convex target sites that employ geometrically matching concave scaffolds. We used this approach to design proteins binding to TGFßRII, CTLA-4 and PD-L1 which following experimental optimization have low nanomolar to picomolar affinities and potent biological activity. Co-crystal structures of the TGFßRII and CTLA-4 binders in complex with the receptors are in close agreement with the design models. Our approach provides a general route to generating very high affinity binders to convex protein target sites.

Living in high-poverty areas is associated with reduced survival in patients with thoracoabdominal aortic aneurysms.

OBJECTIVES: Studies have demonstrated that socioeconomic status, insurance, race, and distance impact clinical outcomes in patients with abdominal aortic aneurysms. The purpose of this study was to assess if these factors also impact clinical outcomes in patients with thoracoabdominal aortic aneurysms (TAAAs). METHODS: We conducted a retrospective review of patients with TAAAs confirmed by computed tomography imaging between 2009 and 2019 at a single institution. Patients' zip codes were mapped to American Community Survey Data to obtain geographic poverty rates. We used the standard U.S. Census definition of high-poverty concentration as >20% of the population living at 100% of the poverty rate. Our primary outcome was overall survival, stratified by whether the patient underwent repair. RESULTS: Of 578 patients, 575 had zip code data and were analyzed. In both the nonoperative (N = 268) and operative (N = 307) groups, there were no significant differences in age, race, comorbidities, clinical urgency, surgery utilization, or surgery modality between patients living in high-poverty areas (N = 95, 16.4%) vs not. In the nonoperative group, patients from high-poverty areas were more likely to have aneurysm due to dissection (37.5% vs 17.6%, P = .03). In multivariate analyses, patients from high-poverty zip codes had significantly worse nonoperative survival (hazard ratio [HR]: 1.9, 95% confidence interval [CI]: 1.1-3.3, P = .03). In the repair group, high poverty was also a significant predictor of reduced postoperative survival (HR: 1.65, 95% CI: 1-2.63, P = .04). Adding the Gagne Index, these differences persisted in both groups (nonoperative: HR: 1.93, 95% CI: 1.01-3.70, P = .05; operative: HR: 1.62, 95% CI: 1.03-2.56, P = .04). In Kaplan-Meier analysis, the difference in postoperative survival began approximately 1.5 years after repair. Private insurance was predictive of improved postoperative survival (HR: 0.42, 95% CI: 0.18-0.95, P = .04) but reduced nonoperative survival (HR: 2.05, 95% 1.01-4.14, P = .04). Data were insufficient to determine if race impacted survival discretely from poverty status. These results were found after adjusting for age, race, sex, maximum aortic diameter, coronary artery disease, distance from the hospital, insurance, and active smoking. Interestingly, in multivariate regression, traveling greater than 100 miles was correlated with increased surgery utilization (odds ratio: 1.58, 95% CI: 1.08-2.33, P = .02) and long-term survival (HR: 0.61, 95% CI: 0.41-0.92, P = .02). CONCLUSIONS: Patients with TAAAs living in high-poverty areas had significantly more dissections and suffered a nearly doubled risk of mortality compared with patients living outside such areas. These data suggest that these disparities are attributed to the overall impacts of poverty and highlight the pressing need for research into TAAA disparities.

De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity.

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in viscoelastic biomaterials exhibiting fluid-like properties under rest and low shear, but solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly in a manner similar to formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.

Accurate computational design of three-dimensional protein crystals.

Protein crystallization plays a central role in structural biology. Despite this, the process of crystallization remains poorly understood and highly empirical, with crystal contacts, lattice packing arrangements and space group preferences being largely unpredictable. Programming protein crystallization through precisely engineered side-chain-side-chain interactions across protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach for designing three-dimensional protein crystals with prespecified lattice architectures at atomic accuracy that hierarchically constrains the overall number of degrees of freedom of the system. We design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into >100 µm three-dimensional crystals. The structures of these crystals are nearly identical to the computational design models, closely corresponding in both overall architecture and the specific protein-protein interactions. The dimensions of the crystal unit cell can be systematically redesigned while retaining the space group symmetry and overall architecture, and the crystals are extremely porous and highly stable. Our approach enables the computational design of protein crystals with high accuracy, and the designed protein crystals, which have both structural and assembly information encoded in their primary sequences, provide a powerful platform for biological materials engineering.

De novo design of modular protein hydrogels with programmable intra- and extracellular viscoelasticity.

Relating the macroscopic properties of protein-based materials to their underlying component microstructure is an outstanding challenge. Here, we exploit computational design to specify the size, flexibility, and valency of de novo protein building blocks, as well as the interaction dynamics between them, to investigate how molecular parameters govern the macroscopic viscoelasticity of the resultant protein hydrogels. We construct gel systems from pairs of symmetric protein homo-oligomers, each comprising 2, 5, 24, or 120 individual protein components, that are crosslinked either physically or covalently into idealized step-growth biopolymer networks. Through rheological assessment and molecular dynamics (MD) simulation, we find that the covalent linkage of multifunctional precursors yields hydrogels whose viscoelasticity depends on the crosslink length between the constituent building blocks. In contrast, reversibly crosslinking the homo-oligomeric components with a computationally designed heterodimer results in non-Newtonian biomaterials exhibiting fluid-like properties under rest and low shear, but shear-stiffening solid-like behavior at higher frequencies. Exploiting the unique genetic encodability of these materials, we demonstrate the assembly of protein networks within living mammalian cells and show via fluorescence recovery after photobleaching (FRAP) that mechanical properties can be tuned intracellularly, in correlation with matching formulations formed extracellularly. We anticipate that the ability to modularly construct and systematically program the viscoelastic properties of designer protein-based materials could have broad utility in biomedicine, with applications in tissue engineering, therapeutic delivery, and synthetic biology.

Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies.

Growth factors and cytokines signal by binding to the extracellular domains of their receptors and drive association and transphosphorylation of the receptor intracellular tyrosine kinase domains, initiating downstream signaling cascades. To enable systematic exploration of how receptor valency and geometry affects signaling outcomes, we designed cyclic homo-oligomers with up to 8 subunits using repeat protein building blocks that can be modularly extended. By incorporating a de novo designed fibroblast growth-factor receptor (FGFR) binding module into these scaffolds, we generated a series of synthetic signaling ligands that exhibit potent valency- and geometry-dependent Ca2+ release and MAPK pathway activation. The high specificity of the designed agonists reveal distinct roles for two FGFR splice variants in driving endothelial and mesenchymal cell fates during early vascular development. The ability to incorporate receptor binding domains and repeat extensions in a modular fashion makes our designed scaffolds broadly useful for probing and manipulating cellular signaling pathways.

Single Cell RNA Sequencing Reveals Human Tooth Type Identity and Guides In Vitro hiPSC Derived Odontoblast Differentiation (iOB).

Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Application of this sci-RNA-seq analysis generates a differentiation protocol to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and its biomineralization capacity using the de novo designed FGFR1/2c isoform specific minibinder scaffolded as a C6 oligomer that acts as a pathway agonist. We find that FGFR1c is upregulated in functional odontoblasts and specifically plays a crucial role in driving odontoblast maturity. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Women with thoracoabdominal aortic aneurysms have increased frailty and more complex aortic anatomy compared with men.

OBJECTIVE: Operative repair of thoracoabdominal aortic aneurysms (TAAAs) is high risk, and many patients will be unfit for intervention. Prior studies have noted lower rates of repair for women than for men. The reasons for this disparity have remained unknown but could include a greater burden of co-morbid illness or anatomic barriers. Frailty could also contribute to the lower intervention rates but has rarely been reported in preoperative risk assessments. The aim of the present study was to assess the sex-related differences in clinical comorbidities, anatomic suitability, and frailty among an unselected cohort of patients who had presented with TAAAs. METHODS: All patients with extent I to V TAAAs confirmed by computed tomography imaging between 2009 and 2019 at a single institution were reviewed. Patients were included regardless of whether they had undergone repair. Clinical comorbidities, anatomic details, and metrics of frailty were collected and used to determine operative risk. RESULTS: Of the 578 identified patients, 233 (40%) were women. The women were older than the men at diagnosis (71 years vs 68 years; P = .006) but had had similar comorbidities, with the exception of lower rates of coronary artery disease (37% vs 47%; P = .04) and higher rates of chronic obstructive pulmonary disease (45% vs 36%; P = .008). The Society for Vascular Surgery clinical comorbidity score was similar between the sexes. Women were less likely to have undergone prior aortic surgery (32% vs 53%; P < .0001) but had had more extensive aneurysms (P = .007) with greater rates of prohibitive anatomic risk factors (open repair, 31% vs 17% [P = .01]; endovascular repair, 33% vs 28% [P = .32]). The metrics of frailty were higher for the women, including recent unintentional weight loss (11% vs 5%; P = .002), limited physical activity tolerance (46% vs 31%; P < .0001), and the need for ambulatory assistance (13% vs 6%; P < .0001). Of the 578 patients, 55% of the women and 30% of the men had had at least one frailty metric that was prohibitive for open repair (P = .0006). The women had also scored higher on the modified frailty index (P = .009). For open repair, 74% of women and 61% of men had at least one prohibitive risk factor. The women were also more likely to have multiple types of prohibitive risk factors. Compared with the men, the women were less likely to be offered repair (60% vs 74%; P = .0009) and less likely to undergo repair (44% vs 62%; P = .0001). CONCLUSIONS: Women with TAAAs had increased metrics of frailty and anatomic risk that were not captured by comorbidity-based risk assessments. This suggests that frailty, together with complex anatomy, could explain the lower intervention rates for women with TAAAs.

Reconfigurable asymmetric protein assemblies through implicit negative design.

Asymmetric multiprotein complexes that undergo subunit exchange play central roles in biology but present a challenge for design because the components must not only contain interfaces that enable reversible association but also be stable and well behaved in isolation. We use implicit negative design to generate ß sheet-mediated heterodimers that can be assembled into a wide variety of complexes. The designs are stable, folded, and soluble in isolation and rapidly assemble upon mixing, and crystal structures are close to the computational models. We construct linearly arranged hetero-oligomers with up to six different components, branched hetero-oligomers, closed C4-symmetric two-component rings, and hetero-oligomers assembled on a cyclic homo-oligomeric central hub and demonstrate that such complexes can readily reconfigure through subunit exchange. Our approach provides a general route to designing asymmetric reconfigurable protein systems.

Multivalent designed proteins protect against SARS-CoV-2 variants of concern.

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC 50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. ONE-SENTENCE SUMMARY: We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

Design of multi-scale protein complexes by hierarchical building block fusion.

A systematic and robust approach to generating complex protein nanomaterials would have broad utility. We develop a hierarchical approach to designing multi-component protein assemblies from two classes of modular building blocks: designed helical repeat proteins (DHRs) and helical bundle oligomers (HBs). We first rigidly fuse DHRs to HBs to generate a large library of oligomeric building blocks. We then generate assemblies with cyclic, dihedral, and point group symmetries from these building blocks using architecture guided rigid helical fusion with new software named WORMS. X-ray crystallography and cryo-electron microscopy characterization show that the hierarchical design approach can accurately generate a wide range of assemblies, including a 43 nm diameter icosahedral nanocage. The computational methods and building block sets described here provide a very general route to de novo designed protein nanomaterials.

Sex-related outcomes after fenestrated-branched endovascular aneurysm repair for thoracoabdominal aortic aneurysms in the U.S. Fenestrated and Branched Aortic Research Consortium.

OBJECTIVE: Fenestrated-branched endovascular aneurysm repair (FBEVAR) has expanded the treatment of patients with thoracoabdominal aortic aneurysms (TAAAs). Previous studies have demonstrated that women are less likely to be treated with standard infrarenal endovascular aneurysm repair because of anatomic ineligibility and experience greater mortality after both infrarenal and thoracic aortic aneurysm repair. The purpose of the present study was to describe the sex-related outcomes after FBEVAR for treatment of TAAAs. METHODS: The data from 886 patients with extent I to IV TAAAs (excluding pararenal or juxtarenal aneurysms), enrolled in eight prospective, physician-sponsored, investigational device exemption studies from 2013 to 2019, were analyzed. All data were collected prospectively, audited and adjudicated by clinical events committees and/or data safety monitoring boards, and subject to Food and Drug Administration oversight. All the patients had been treated with Cook-manufactured patient-specific FBEVAR devices or the Cook t-Branch off-the-shelf device (Cook Medical, Brisbane, Australia). RESULTS: Of the 886 patients who underwent FBEVAR, 288 (33%) were women. The women had more extensive aneurysms and a greater prevalence of diabetes (33% vs 26%; P = .043) but a lower prevalence of coronary artery disease (33% vs 52%; P < .0001) and previous infrarenal endovascular aneurysm repair (7.6% vs 16%; P < .001). The women had required a longer operative time from incision to surgery end (5.0 ± 1.8 hours vs 4.6 ± 1.7 hours; P < .001), experienced lower technical success (93% vs 98%; P = .002), and were less likely to be discharged to home (72% vs 83%; P = .009). Despite the smaller access vessels, the women did not have an increased incidence of access site complications. Also, the 30-day outcomes were broadly similar between the sexes. At 1 year, no differences were found between the women and men in freedom from type I or III endoleak (91.4% vs 92.0%; P = .64), freedom from reintervention (81.7% vs 85.3%; P = .10), target vessel instability (87.5% vs 89.2%; P = .31), and survival (89.6% vs 91.7%; P = .26). The women had a greater incidence of postoperative sac expansion (12% vs 6.5%; P = .006). Multivariable modeling adjusted for age, aneurysm extent, aneurysm size, urgent procedure, and renal function showed that patient sex was not an independent predictor of survival (hazard ratio, 0.83; 95% confidence interval, 0.50-1.37; P = .46). CONCLUSIONS: Women undergoing FBEVAR demonstrated metrics of increased complexity and had a lower level of technical success, especially those with extensive aneurysms. Compared with the men, the women had similar 30-day mortality and 1-year outcomes, with the exception of an increased incidence of sac expansion. These data have demonstrated that FBEVAR is safe and effective for women and men but that further efforts to improve outcome parity are indicated.

Anatomic Eligibility for Commercial Branched Endograft Repair of Thoracoabdominal Aortic Aneurysms.

BACKGROUND: First-generation "off-the-shelf" branched endovascular stent grafts are in development for treatment of thoracoabdominal aortic aneurysms (TAAAs). Prior studies have assessed eligibility rates among highly selected cohorts of patients referred for endovascular treatment, and the broader applicability of these devices to all patients with TAAA is unknown. The aims of this study were to assess the overall suitability of the 3 commercial 4-branched devices with or without adjunct procedure(s) in an unselected cohort of patients with TAAA and to identify areas for improvement in the next generation of devices. METHODS: A retrospective review of three-dimensional centerline reconstructions of contrast-enhanced computed tomography (CT) imaging was performed in consecutive patients with TAAA seen between 2013 and 2017. All patients with contrast-enhanced CT imaging were included, regardless of prior evaluation for suitability for endovascular repair. Eligibility for a device was assessed based on instructions for use (IFU) from the device manufacturer along with prespecified anatomic criteria. Adjunct procedures were defined as carotid-subclavian revascularization, target vessel endovascular intervention, and iliac conduit/revascularization. RESULTS: Of 165 patients with TAAA, 122 had CT scans adequate for study inclusion. Eighteen patients (14.8%) were eligible for at least 1 device by IFU, and 41 (33.6%) could have been made eligible for at least 1 device by an adjunct procedure. Sixty-three (51.6%) were not eligible for any device within IFU even with adjunct procedures, including 31 of 32 patients with TAAA due to dissection. The most common reasons for ineligibility were perivisceral flow channel diameter <20 mm (n = 43) and an inadequate proximal seal zone (n = 29). Women were significantly less likely to be eligible for an off-the-shelf device (P = 0.03) and were more likely to require an iliac procedure to become eligible (P = 0.006). Almost none of the patients with dissection could receive a device even if adjunct procedures were used. CONCLUSIONS: Over half of patients with TAAA could not be made eligible for an off-the-shelf device based on manufacturers' criteria, even with adjunct procedures. Women and patients with TAAA due to dissection had higher rates of ineligibility. These data demonstrate that custom fenestrated devices and low-profile devices are needed to expand eligibility for endovascular repair of TAAA.

Genetic analysis of the CDI pathway from Burkholderia pseudomallei 1026b.

Contact-dependent growth inhibition (CDI) is a mode of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion systems. CdiA binds to receptors on susceptible target bacteria, then delivers a toxin domain derived from its C-terminus. Studies with Escherichia coli suggest the existence of multiple CDI growth-inhibition pathways, whereby different systems exploit distinct target-cell proteins to deliver and activate toxins. Here, we explore the CDI pathway in Burkholderia using the CDIIIBp1026b system encoded on chromosome II of Burkholderia pseudomallei 1026b as a model. We took a genetic approach and selected Burkholderia thailandensis E264 mutants that are resistant to growth inhibition by CDIIIBp1026b. We identified mutations in three genes, BTH_I0359, BTH_II0599, and BTH_I0986, each of which confers resistance to CDIIIBp1026b. BTH_I0359 encodes a small peptide of unknown function, whereas BTH_II0599 encodes a predicted inner membrane transport protein of the major facilitator superfamily. The inner membrane localization of BTH_II0599 suggests that it may facilitate translocation of CdiA-CTIIBp1026b toxin from the periplasm into the cytoplasm of target cells. BTH_I0986 encodes a putative transglycosylase involved in lipopolysaccharide (LPS) synthesis. ∆BTH_I0986 mutants have altered LPS structure and do not interact with CDI⁺ inhibitor cells to the same extent as BTH_I0986⁺ cells, suggesting that LPS could function as a receptor for CdiAIIBp1026b. Although ∆BTH_I0359, ∆BTH_II0599, and ∆BTH_I0986 mutations confer resistance to CDIIIBp1026b, they provide no protection against the CDIE264 system deployed by B. thailandensis E264. Together, these findings demonstrate that CDI growth-inhibition pathways are distinct and can differ significantly even between closely related species.