SummaryProton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug absorption. These transporters accept a wide range of substrates, but the structural basis for how different peptide side chains are accommodated has so far remained obscure. Twenty-eight peptides were screened for binding to PepTSt from Streptococcus thermophilus, and structures were determined of PepTSt in complex with four physicochemically diverse dipeptides, which bind with millimolar affinity: Ala-Leu, Phe-Ala, Ala-Gln, and Asp-Glu. The structures show that PepTSt can adapt to different peptide side chains through movement of binding site residues and water molecules, and that a good fit can be further aided by adjustment of the position of the peptide itself. Finally, structures were also determined in complex with adventitiously bound HEPES, polyethylene glycol, and phosphate molecules, which further underline the adaptability of the binding site.

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

41The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic 42 neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. 43 However, traditional antibody production is hampered by long development times and costly 44 production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic 45 library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-46 2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity 47 were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 48 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively 49 in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation 50 of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach 51 represents an alternative, fast workflow to select binders with neutralizing activity against newly 52 emerging viruses. 53 54 55 56 57 58 59 60 61The emerging COVID-19 pandemic imposes a substantial social and economic burden worldwide. 62The causative agent of COVID-19 that causes, among other symptoms, severe atypical pneumonia 63 in humans was quickly identified as a novel coronavirus and designated as SARS-CoV-2 1-4 . 64Similar to its closest homolog SARS-CoV-1, SARS-CoV-2 exploits the ACE2 (angiotensin 65 converting enzyme 2) receptor to enter host cells [5][6][7] . Host cell entry is orchestrated by the viral 66 spike protein that, upon binding to ACE2, mediates fusion of the viral membrane with the host 67 membrane. The ectodomain of the heavily glycosylated spike protein forms a trimer where each 68 protomer consists of a core S2 subunit and a distal S1 subunit. Each S1 subunit comprises a 69 receptor binding domain (RBD) that can switch between an exposed 'up' conformation and a 70 'down' conformation, where the latter is inaccessible for ACE2 binding [8][9][10] . A number of 71 antibodies against various viral surface proteins are currently in clinical trials 11 . Exposed on the 72 surface of SARS-CoV-2, the spike protein is the major antigenic determinant of the host immune 73 response and one of the main coronavirus drug targets 12,13 . Indeed, plasma from convalescent 74 SARS-CoV-2 patients was suggested to improve the clinical outcome of patients with severe 75 COVID-19 [14][15][16] . Several groups have isolated human antibodies that showed promising 76 neutralization activity against SARS-CoV-2 in vitro and improved the clinical outcome of tested 77 animals in vivo [17][18][19][20] . As an alternative to human antibodies, nanobodies (single-domain antibodies) 78 can also be used as therapeutics with the advantage of their small size, increased stability and 79 superior simplicity in production 21,22 . Recently, several groups showed that nanobodies can inhibit 80 the binding of the spike protein to ACE2 and neutralize the virus ...